Compositions comprising modified HIV envelopes

ABSTRACT

The invention is directed to immunogens and methods for inducing immune responses, comprising methods for germline B cell stimulation and maturation by reverse engineering of HIV-1 envelopes.

This application is a U.S. National Stage application under 35 U.S.C. §371 of International Patent Application No. PCT/US2018/34772, filed onMay 25, 2018, which claims the benefit of and priority to U.S.Provisional Application No. 62/511,226 filed May 25, 2017 and U.S.Provisional Application No. 62/565,952 filed Sep. 29, 2017, the contentsof each of which are hereby incorporated by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Apr. 10, 2020, is named1234300 00334US2 SL.txt and is 1,617,443 bytes in size.

TECHNICAL FIELD

The present invention relates in general, to a composition suitable foruse in inducing anti-HIV-1 antibodies, and, in particular, toimmunogenic compositions comprising envelope proteins and nucleic acidsto induce cross-reactive neutralizing antibodies and increase theirbreadth of coverage. The invention also relates to methods of inducingsuch broadly neutralizing anti-HIV-1 antibodies using such compositions.

BACKGROUND

The development of a safe and effective HIV-1 vaccine is one of thehighest priorities of the scientific community working on the HIV-1epidemic. While anti-retroviral treatment (ART) has dramaticallyprolonged the lives of HIV-1 infected patients, ART is not routinelyavailable in developing countries.

SUMMARY OF THE INVENTION

The ability to stimulate germline B cells that give rise to broadlyneutralizing antibodies (bNAbs) is a major goal for HIV-1 vaccines.BNAbs that target the CD4-binding site (CD4bs) of HIV-1 and exhibitextraordinary potency and breadth of neutralization are particularlyattractive to elicit with vaccines. Glycans that border the CD4bs andimpede the binding of germline-reverted forms of CD4bs bNAbs arepotential barriers to naïve B cell receptor engagement. In some aspects,pseudovirus neutralization was used as a means to identify Envmodifications that permit native Env trimer binding to germline revertedCD4bs bNAb CH235.12 (VH1-46) as a surrogate for naïve B cell receptorengagement.

Site-directed mutagenesis was used to create strategic mutants ofautologous CH0505TF Env. The mutants were produced in cells lacking theenzyme N-acetylglucosaminyltransferase (GnTI-) to enrich for Man5glycoforms of N-linked glycans that would otherwise be fully processedinto complex-type glycans. Naturally-glycosylated and Man5-enrichedforms of parental and mutant Envs were tested for neutralization by theCH235 antibody lineage that included the unmutated common ancestor(UCA), intermediates and mature forms of CH235.12. CorrespondingSOSIP.664 trimers were tested for UCA binding. These strategies are usedto create germline-targeting and reverse-engineered immunogens to ElicitCH235.12 Lineage BNAbs.

In one aspect the invention provides that Man5-enriched CH0505TFcontaining two VRC01-class resistance mutations, N279K (loop D) andG458Y (V5 region), was highly susceptible to neutralization by CH235UCA. This double mutant was also neutralized by the UCA when produced in293T cells but was 100× more sensitive when produced in GnTI-cells(Man5-enrichment). Neutralization predicted nM affinity binding tovarious envelopes, e.g. but not limited to mutated, Man5-enrichedCH0505TF SOSIP.664 trimers.

In one aspect the invention provides recombinant HIV-1 envelopepolypeptides from Tables 1A-B, Examples 2-13, or any other envelope,wherein the envelope comprises G458Mut. In some embodiments, optionallythe polypeptide is enriched for Man5 glycoforms of N-linked glycans. Incertain embodiments G458Mut is G458Y. In certain embodimentsnon-limiting embodiments of G458Mut are described in Ex. 10 and FIG. 89. In the embodiments where the polypeptide is not enriched for Man5glycoforms of N-linked glycans, the polypeptide is recombinantlyproduced in any suitable cell line wherein the polypeptide is fullyglycosylated compared to the enrichment for Man5 glycoforms of N-linkedglycans in GnTI−/− cells. In one embodiment, 293T cells are used toproduce fully or naturally glycosylated polypeptides. Other cells may beused in place of 293T cells to produce naturally or fully glycosylatedimmunogens.

In one aspect, the invention provides a recombinant HIV-1 envelopepolypeptide from Tables 1A-B, Examples 2-13, or any other envelope,wherein the polypeptide is enriched for Man5 glycoforms of N-linkedglycans, and wherein in some embodiments the polypeptide hasdifferential binding and/neutralization compared to fully glycosylatedenvelope.

In one aspect the invention provides a nucleic acid encoding therecombinant polypeptides of the invention.

In one aspect the invention provides a recombinant trimer comprisingthree identical protomers of an HIV-1 envelope polypeptide of theinvention. In one aspects the invention provides an immunogeniccomposition comprising the recombinant trimer of the invention and acarrier, wherein the trimer comprises three identical protomers of anHIV-1 envelope polypeptide. In certain embodiments, the compositioncomprises which are substantially homogenous.

In one aspect the invention provides an immunogenic compositioncomprising nucleic acid encoding the recombinant HIV-1 envelopepolypeptide of the invention and a carrier.

In certain embodiments, the recombinant HIV-1 envelope polypeptide isHIV-1 CH505 M5. In certain embodiments, the recombinant HIV-1 envelopepolypeptide is HIV-1 CH505 T/F. In certain embodiments, the recombinantHIV-1 envelope polypeptide is HIV-1 CH505 M11.

In one aspect the invention provides methods of using the immunogens ofthe invention to induce immune response, wherein in some embodimentswithout limitation these immune responses stimulate germline B cellsthat give rise to broadly neutralizing antibodies (bNAbs). Non-limitingembodiments of methods are described in FIGS. 58A and 58B. In one aspectthe invention provides methods of inducing an immune response in asubject comprising administering a composition comprising any suitableform of an HIV-1 envelope(s) in an amount sufficient to induce an immuneresponse from one or more of the envelopes of the preceding paragraphs,wherein in one embodiment the envelope is: (a) envelope CH505 M5G458Mut, (b) envelope CH505 M5, wherein the envelope is enriched forMan5 glycoforms of N-linked glycans; (c) CH 505 M5 G458Mut, wherein theenvelope is enriched for Man5 glycoforms of N-linked glycans, or anycombination thereof.

Any one of the methods of the invention wherein the administration stepcan alternatively, or in addition, comprise administering a nucleic acidencoding the corresponding HIV-1 polypeptide(s) in an amount sufficientto induce an immune response.

Any one of the methods of the invention wherein the method comprisesadministering a composition comprising HIV-1 envelope CH505 M5 G458Mutor a nucleic acid encoding HIV-1 envelope CH505 M5 G458Mut.

Any one of the methods of the invention further comprising administeringa composition comprising HIV-1 envelope CH505 M5, wherein the envelopeis enriched for Man5 glycoforms of N-linked glycans.

Any one of the methods of the invention further comprising administeringa composition comprising HIV-1 envelope CH505 M5 G458Mut, wherein theenvelope is enriched for Man5 glycoforms of N-linked glycans wherein theenvelope is enriched for Man5 glycoforms of N-linked glycans.

Any one of the methods of the invention wherein the method comprisesadministering a composition comprising HIV-1 envelope CH505 M5 G458Mut,wherein the envelope is enriched for Man5 glycoforms of N-linkedglycans.

Any one of the methods of the invention further comprising administeringa composition comprising CH505 M5, wherein the envelope is enriched forMan5 glycoforms of N-linked glycans

Any one of the methods of the invention further comprises administeringa composition comprising HIV-1 envelope CH505 M5 G458Mut or a nucleicacid encoding HIV-1 envelope CH505 M5 G458Mut.

Any one of the methods of the invention further comprising administeringa composition comprising HIV-1 envelope CH505 T/F, wherein the envelopeis enriched for Man5 glycoforms of N-linked glycans.

Any one of the methods of the invention further comprising administeringa composition comprising HIV-1 envelope CH505 M5, wherein the envelopeis enriched for Man5 glycoforms of N-linked glycans and HIV-1 envelopeCH505 M5 G458Mut, wherein the envelope is enriched for Man5 glycoformsof N-linked glycans.

Any one of the methods of the invention further comprising administeringa composition comprising HIV-1 envelope CH505 M5 and HIV-1 envelopeCH505 M5 G458Mut.

Any one of the methods of the invention further comprising administeringa composition comprising HIV-1 envelope CH505 T/F, wherein the envelopeis enriched for Man5 glycoforms of N-linked glycans.

Any one of the methods of the invention further comprising administeringa composition comprising HIV-1 envelope CH 505 T/F.

Any one of the methods of the invention wherein the polypeptide is gp120envelope, gp120D8 envelope, a gp140 envelope (gp140C, gp140CF, gp140CFI)as soluble or stabilized protomer of a SOSIP trimer, a gp145 envelope, agp150 envelope, or a transmembrane bound envelope.

Any one of the methods or compositions of the invention wherein thecomposition further comprises an adjuvant.

Any one of the methods of the invention further comprising administeringan agent which modulates host immune tolerance.

Any one of the methods or compositions of the invention wherein thepolypeptide administered is multimerized. Non-limiting embodiments ofmultimerized envelopes include ferritin particles, liposomes,nanoparticles, or any other suitable form.

Any one of the methods of the invention further comprising administeringan additional immunogen. Non-limiting embodiments are described Example2-13.

In some aspects, these findings advance our understanding of therestrictions imposed by glycans in the elicitation of CD4bs bNAbs andprovide a conceptual framework and methods for immunogen design toinitiate and mature the CH235.12 bNAb lineage.

In one aspect, the invention is directed to immunogens and methods forgermline B cell stimulation and maturation by reverse engineering ofHIV-1 envelopes. B cell stimulation is a key initial step in the abilityof HIV vaccines to elicit broadly neutralizing antibodies (bNAbs). Insome aspects the invention provides modifications of HIV-1 envelopes totrigger germline activation and drive subsequent B cell maturation ofbNAbs, including but not limited to CD4bs bNAbs.

In certain aspects, the invention is directed to a recombinant HIV-1envelope polypeptide, including but not limited to an envelope fromTables 1A-B, wherein the envelope comprises G458Mut and/or glycosylationpattern similar to the glycosylation patter of an envelope grown inGnTI^(−/−) cells. In certain embodiments, the polypeptide is anon-naturally occurring protomer designed to form an envelope trimer.The glycosylation pattern of GnTI−/− grown recombinant polypetides iswell known. In some embodiments, when produced in GnTI−/− cells thepolypeptides are enriched for Man5 glycoforms of N-linked glycans.

In certain aspects, the invention provides nucleic acids encoding theserecombinant polypeptides. In certain aspects, the invention providesrecombinant cells and/or population of recombinant cells comprisingnucleic acids encoding the recombinant polypeptides of the invention.

In certain embodiments, the invention provides a recombinant trimercomprising three identical protomers of an envelope from Tables 1A-B. Incertain embodiments, the invention provides an immunogenic compositioncomprising the recombinant trimer and a carrier, wherein the trimercomprises three identical protomers of an HIV-1 envelope listed inTables 1A-B.

In certain embodiments, the invention provides an immunogeniccomposition comprising nucleic acid encoding a recombinant HIV-1envelope and a carrier. The compositions could comprise an adjuvant.

In certain embodiments the recombinant envelope is HIV-1 envelope CH505M5 or a nucleic acid encoding HIV-1 envelope CH505M5, wherein the HIV-1CH505 M5 envelope comprises a G458Mut and is recombinantly produced in293T cells so that glycosylation pattern is not Man5 enriched. Incertain embodiments the recombinant envelope is HIV-1 envelope CH505 M5or a nucleic acid encoding HIV-1 envelope CH505 M5, wherein the HIV-1CH505 M5 envelope does not comprise a G458Mut and is recombinantlyproduced in GnTI−/− cells so that glycosylation pattern is Man5enriched. In certain embodiments the recombinant envelope is HIV-1envelope CH505 M5 or a nucleic acid encoding HIV-1 envelope CH505 M5,wherein the HIV-1 CH505 M5 envelope comprises a G458Mut and isrecombinantly produced in GnTI−/− cells so that glycosylation pattern isMan5 enriched.

In certain embodiments the recombinant envelope is HIV-1 envelope CH505M11 or a nucleic acid encoding HIV-1 envelope CH505 M11, wherein theHIV-1 CH505 M11 envelope comprises a G458Mut and is recombinantlyproduced in 293T cells so that glycosylation pattern is not Man5enriched. In certain embodiments the recombinant envelope is HIV-1envelope CH505 M11 or a nucleic acid encoding HIV-1 envelope CH505 M11,wherein the HIV-1 M11 envelope does not comprise a G458Mut and isrecombinantly produced in GnTI−/− cells so that glycosylation pattern isMan5 enriched. In certain embodiments the recombinant envelope is HIV-1envelope CH505 M11 or a nucleic acid encoding HIV-1 envelope CH505 M11,wherein the HIV-1 CH505 M11 envelope comprises a G458Mut and isrecombinantly produced in GnTI−/− cells so that glycosylation pattern isMan5 enriched.

In certain embodiments the recombinant envelope is HIV-1 envelope CH505T/F or a nucleic acid encoding HIV-1 envelope CH505 T/F, wherein theHIV-1 CH505 T/F envelope comprises a G458Mut and is recombinantlyproduced in 293T cells so that glycosylation pattern is not Man5enriched. In certain embodiments the recombinant envelope is HIV-1envelope CH505 T/F or a nucleic acid encoding HIV-1 envelope CH505 T/F,wherein the HIV-1 CH505 T/F envelope does not comprise a G458Mut and isrecombinantly produced in GnTI−/− cells so that glycosylation pattern isMan5 enriched. In certain embodiments the recombinant envelope is HIV-1envelope CH505 T/F or a nucleic acid encoding HIV-1 envelope v, whereinthe HIV-1 CH505 T/F 5 envelope comprises a G458Mut and is recombinantlyproduced in GnTI−/− cells so that glycosylation pattern is Man5enriched.

In certain aspects the invention provides methods of inducing immuneresponses using the inventive immunoges. In one embodiment the inventionprovides a method of inducing an immune response in a subject comprisingadministering a composition in an amount sufficient to induce an immuneresponse, wherein the composition comprises any suitable form of anucleic acid(s) encoding an HIV-1 envelope(s) from one or more of thefollowing groups:

-   -   (a) envelopesCH505 M5, M11, w20.14, w30.20, w30.12, and w136.B18        (Selection F, e.g. listed in FIG. 18A) or any combination        thereof;    -   (b) envelopes CH505 M5, w30.25, w53.25, and w53.29 (Selection G,        e.g. FIG. 20 ) or any combination thereof;    -   (c) envelopes CH505 M5, w30.20, w20.14, and w30.12 (Selection H,        e.g. FIG. 21 ) or any combination thereof,    -   and wherein the administration step can alternatively, or in        addition, comprise administering an HIV-1 polypeptide(s) in an        amount sufficient to induce an immune response from one or more        of the following groups:    -   (a) envelopes CH505 M5, M11, w20.14, w30.20, w30.12, and        w136.B18 (Selection F, e.g. listed in FIG. 18A) or any        combination thereof;    -   (b) envelopes CH505 M5, w30.25, w53.25, and w53.29 (Selection G,        e.g. FIG. 20 ) or any combination thereof;    -   (c) envelopes CH505 M5, w30.20, w20.14, and w30.12 (Selection H,        e.g. FIG. 21 ) or any combination thereof;    -   Wherein in some embodiments the envelopes comprise G458Mut        and/or in some embodiments have glycosylation pattern similar to        the glycosylation patter of an envelope grown in GnTI^(−/−)        cells.

In certain embodiments the methods comprise administering immunogenswith increasing BCR stimulation (See e.g. FIG. 58 ). In certainembodiments the methods comprise administering recombinant HIV-1envelope CH505 M5 or a nucleic acid encoding HIV-1 envelope CH505M5,wherein the HIV-1 CH505 M5 envelope comprises a G458Mut and isrecombinantly produced in 293T cells so that glycosylation pattern isnot Man5 enriched. In certain embodiments the methods comprising therecombinant envelope is HIV-1 envelope CH505 M5 or a nucleic acidencoding HIV-1 envelope CH505 M5, wherein the HIV-1 CH505 M5 envelopedoes not comprise a G458Mut and is recombinantly produced in GnTI−/−cells so that glycosylation pattern is Man5 enriched. In certainembodiments the methods comprise administering the recombinant envelopeis HIV-1 envelope CH505 M5 or a nucleic acid encoding HIV-1 envelopeCH505 M5, wherein the HIV-1 CH505 M5 envelope comprises a G458Mut and isrecombinantly produced in GnTI−/− cells so that glycosylation pattern isMan5 enriched.

In some embodiments the methods further comprise administering HIV-1envelope w20.14 or a nucleic acid encoding HIV-1 envelope w20.14,followed by administering HIV-1 envelope w30.20 or a nucleic acidencoding HIV-1 envelope w30.20, and followed by administering HIV-1envelope w30.12 or a nucleic acid encoding HIV-1 envelope w30.12.

In some embodiments the methods further comprise administering HIV-1envelope w136.B18 or a nucleic acid encoding HIV-1 envelope w136.B18.

In some embodiments the methods further comprise administering HIV-1envelope w30.25 or a nucleic acid encoding HIV-1 envelope w30.25, HIV-1envelope w53.25 or a nucleic acid encoding HIV-1 envelope w53.25, HIV-1envelope w53.29 or a nucleic acid encoding HIV-1 envelope w53.29.

A method of inducing an immune response in a subject comprisingadministering a composition in an amount sufficient to induce an immuneresponse, wherein the composition comprises any suitable form of anucleic acid(s) encoding an HIV-1 envelope(s) in an amount sufficient toinduce an immune response from one or more of the following groups:

-   -   (a) envelopes CH 505 T/F, M5, w53.16, w78.33, and w100.B6 or any        combination thereof; wherein the envelopes comprise G458Mut,    -   and wherein the administration step can alternatively, or in        addition, comprise administering an HIV-1 polypeptide(s) in an        amount sufficient to induce an immune response from one or more        of the following groups:    -   (a) envelopes CH 505 T/F, M5, w53.16, w78.33, and w100.B6 or any        combination thereof; wherein the envelopes comprise G458Mut        and/or glycosylation pattern similar to the glycosylation patter        of an envelope grown in GnTI^(−/−) cells.

In certain embodiments, the invention provides compositions and methodfor induction of immune response, for example cross-reactive (broadly)neutralizing Ab induction. In certain embodiments, the methods usecompositions comprising “swarms” of sequentially evolved envelopeviruses that occur in the setting of bnAb generation in vivo in HIV-1infection.

In certain aspects the invention provides modified HIV envelopes,wherein the modified envelopes are suitable for use as immunogens forgermline targeting of CD4bs broadly neutralizing antibodies. In certainaspects, the modified envelopes of the invention could be used in assaysto determine whether CD4bs broad neutralization antibodies lineage(s)have been induced by vaccine regimens.

In certain aspects the invention provides compositions comprising aselection of HIV-1 envelopes and/or nucleic acids encoding theseenvelopes as described herein for example but not limited to Selectionsas described herein. Without limitations, these selected combinationscomprise envelopes which provide representation of the sequence(genetic) and antigenic diversity of the HIV-1 envelope variants whichlead to the induction and maturation of the CH103 and CH235 antibodylineages. In certain embodiments the selections of envelopes compriseenvelopes which show differential binding to an antibody or antibodiesfrom CH103 and CH235 lineages (FIGS. 14-16 , FIGS. 17-24 , FIG. 59 ,FIGS. 80-82 ). Non-limiting embodiments of various selections ofimmunogens are described in Example 3 and Examples 10-13. In certainembodiments, these compositions are used in immunization methods as aprime and/or boost. The immunogens could be administered as nucleicacids, polypeptides and/or combination.

In certain embodiments the selections of envelopes comprise envelopeswhich show differential binding to an antibody or antibodies from CH103and CH235 lineages (FIGS. 14-16 , FIGS. 17-24 , FIG. 59 , FIGS. 80-82 ).In certain aspects, the invention provides a kit comprising acombination/selection of immunogens of Example 3, Examples 10-13. Insome embodiments the selection of immunogens is selection F, selectionG, or selection H. In some embodiments the kit comprises instructions onhow to carry out the immunization regimen. In some embodiments the kitcomprises instructions on administration of the selection of immunogensas a prime or boost as part of a prime/boost immunization regimen. Incertain aspects, the invention provides a kit comprising any one of theimmunogens of Example 3 and Example 10, or in selection F, G, or H andinstructions on how to carry out an immunization regimen with theimmunogen of the kit, including which immunogen(s) are a primeimmunization and which immunogen(s) comprise a boost immunization. Insome embodiments the kit comprises instructions on administration of theimmunogen as a prime or as a boost as part of a prime/boost immunizationregimen. In some embodiments the immunogen could be administeredsequentially or additively.

In certain aspects, the invention provides a kit comprising Env M5, M11,20.14, 30.20, 30.12, 30.21 30.23, 30.25, 30.28, 53.25, 53.29, 53.31,78.15, 100.B6 and/or 136.B18. In some embodiments the kit comprisesinstructions on how to carry out the immunization regimen, includingwhich immunogen(s) are a prime immunization and which immunogen(s)comprise a boost immunization. In some embodiments the kit comprisesinstructions on administration of the immunogen as a prime or as a boostas part of a prime/boost immunization regimen.

In some embodiments, the kit comprises Env M5, M11, w20.14, w30.20and/or w136.B18 and instructions on administration of the immunogen as aprime or boost as part of a prime/boost immunization regimen with M5,M11, w20.14, w30.20 and/or w136.B18, including which immunogen(s) are aprime immunization and which immunogen(s) comprise a boost immunizationIn some embodiments, the kit comprises Env M5, w30.25, w53.25 and/orw53.29 and instructions on administration of the immunogen as a prime orboost as part of a prime/boost immunization regimen with M5, w30.25,w53.25 and/or w53.29, including which immunogen(s) are a primeimmunization and which immunogen(s) comprise a boost immunization.

In one aspect the invention provides selections of envelopes fromindividual CH505, which selections can be used in compositions forimmunizations to induce lineages of broad neutralizing antibodies. Incertain embodiments, there is some variance in the immunization regimen;in some embodiments, the selection of HIV-1 envelopes may be grouped invarious combinations of primes and boosts, either as nucleic acids,proteins, or combinations thereof. In certain embodiments thecompositions are pharmaceutical compositions which are immunogenic. Incertain embodiments, the compositions comprise amounts of envelopeswhich are therapeutic and/or immunogenic.

In one aspect the invention provides a composition for a prime boostimmunization regimen comprising any one of the envelopes describedherein, or any combination thereof wherein the envelope is a prime orboost immunogen. In certain embodiments the composition for a primeboost immunization regimen comprises one or more envelopes from FIGS.14-16 , FIGS. 17-24 , FIG. 59 , FIGS. 80-82 , Example 3, or fromimmunogen selection F, selection G, or selection H. In some embodiments,the composition for a prime boost immunization regimen comprises one ormore envelopes M5, M11, 20.14, 30.20, 30.12, 30.21 30.23, 30.25, 30.28,53.25, 53.29, 53.31, 78.15, 100.B6 and/or 136.B18.

In one aspect the invention provides a composition comprising any one ofthe envelopes described herein, or any combination thereof—for examplebut not limited to selections in Examples and FIGS. 14, 15, 16, 17-24 .In other aspects, the invention provides use of these selections ofenvelopes in methods to induce an immune response. In non-limitingembodiments the envelope selections induce antibodies in the CH235lineage, for example antibody CH557, (described in Example 8).

In some embodiments, CH505 M11 Env is administered first as a prime,followed by a mixture of a next group of Envs. In some embodiments,grouping of the envelopes is based on their binding affinity for theantibodies expected to be induced. In some embodiments, grouping of theenvelopes is based on chronological evolution of envelope viruses thatoccurs in the setting of bnAb generation in vivo in HIV-1 infection. Insome embodiments Loop D mutants could be included in either prime and/orboost. In some embodiments, the composition comprises an adjuvant. Insome embodiments, the composition and methods comprise use of agents fortransient modulation of the host immune response.

In one aspect the invention provides a composition comprising nucleicacids encoding HIV-1 envelope which is a loop D mutant, e.g. M11 or anyother suitable D loop mutant or combination thereof, e.g. M11 and M5.

In another aspect the invention provides a method of inducing an immuneresponse in a subject comprising administering a composition comprisingHIV-1 envelope M11 and/or M5 as a prime in an amount sufficient toinduce an immune response, wherein the envelope is administered as apolypeptide or a nucleic acid encoding the same. A method of inducing animmune response in a subject comprising administering a compositioncomprising HIV-1 envelope M11 and M5 as a prime in an amount sufficientto induce an immune response, wherein the envelope is administered as apolypeptide or a nucleic acid encoding the same.

In certain embodiments the methods comprise administering any of theselection listed in Example 3. In certain embodiments the methodscomprise administering Envs M5, M11, 20.14, 30.28, 30.23, and/or136.B18. In certain embodiments the methods comprise administering EnvsM5, M11, 20.14, 30.20, 30.23, and/or 136.B18. In certain embodiments themethods comprise administering Envs M5, M11, 20.14, 30.20, 30.12, and/or136.B18. In certain embodiments the methods comprise administeringenvelopes M5, 30.25, 53.25, and/or 53.29.

In certain embodiments the methods further comprise administering acomposition comprising any one of HIV-1 envelope M11, w020.14, w030.28,w078.15, w053.31 or any combination thereof as a boost, wherein theenvelope is administered as a polypeptide or a nucleic acid encoding thesame.

In certain embodiments the methods comprise administering a compositioncomprising any one of HIV-1 envelope M11, M5, w020.14, w030.28, w078.15,w053.31 or any combination thereof as a boost, wherein the envelope isadministered as a polypeptide or a nucleic acid encoding the same.

In another aspect the invention provides a method of inducing an immuneresponse in a subject comprising administering a composition comprisingHIV-1 envelope M11, M5, w020.14, w030.28, w078.15, w053.16, w030.21,w078.33, w100.B6, w053.31 or any combination thereof as a prime and/orboost in an amount sufficient to induce an immune response, wherein theenvelope is administered as a polypeptide or a nucleic acid encoding thesame.

In certain embodiments, the compositions contemplate nucleic acid, asDNA and/or RNA, or proteins immunogens either alone or in anycombination. In certain embodiments, the methods contemplate genetic, asDNA and/or RNA, immunization either alone or in combination withenvelope protein(s).

In certain embodiments the nucleic acid encoding an envelope is operablylinked to a promoter inserted an expression vector. In certain aspectsthe compositions comprise a suitable carrier. In certain aspects thecompositions comprise a suitable adjuvant.

In certain embodiments the induced immune response includes induction ofantibodies, including but not limited to autologous and/orcross-reactive (broadly) neutralizing antibodies against HIV-1 envelope.Various assays that analyze whether an immunogenic composition inducesan immune response, and the type of antibodies induced are known in theart and are also described herein.

In certain aspects the invention provides an expression vectorcomprising any of the nucleic acid sequences of the invention, whereinthe nucleic acid is operably linked to a promoter. In certain aspectsthe invention provides an expression vector comprising a nucleic acidsequence encoding any of the polypeptides of the invention, wherein thenucleic acid is operably linked to a promoter. In certain embodiments,the nucleic acids are codon optimized for expression in a mammaliancell, in vivo or in vitro. In certain aspects the invention providesnucleic acids comprising any one of the nucleic acid sequences ofinvention. In certain aspects the invention provides nucleic acidsconsisting essentially of any one of the nucleic acid sequences ofinvention. In certain aspects the invention provides nucleic acidsconsisting of any one of the nucleic acid sequences of invention. Incertain embodiments the nucleic acid of the invention, is operablylinked to a promoter and is inserted in an expression vector. In certainaspects the invention provides an immunogenic composition comprising theexpression vector.

In certain aspects the invention provides a composition comprising atleast one of the nucleic acid sequences of the invention. In certainaspects the invention provides a composition comprising any one of thenucleic acid sequences of invention. In certain aspects the inventionprovides a composition comprising at least one nucleic acid sequenceencoding any one of the polypeptides of the invention.

In certain aspects the invention provides a composition comprising atleast one nucleic acid encoding HIV-1 envelope M11, M5, w020.14,w030.28, w078.15, w053.16, w030.21, w078.33, w100.B6, w053.31 or anycombination thereof. Non-limiting examples of combinations are shown inExample 2.

In certain embodiments, the compositions and methods employ an HIV-1envelope as polypeptide instead of a nucleic acid sequence encoding theHIV-1 envelope. In certain embodiments, the compositions and methodsemploy an HIV-1 envelope as polypeptide, a nucleic acid sequenceencoding the HIV-1 envelope, or a combination thereof.

The envelope used in the compositions and methods of the invention canbe a gp160, gp150, gp145, gp140, gp120, gp41, N-terminal deletionvariants as described herein, cleavage resistant variants as describedherein, or codon optimized sequences thereof. In certain embodiments thecomposition comprises envelopes as trimers. In certain embodiments,envelope proteins are multimerized, for example trimers are attached toa particle such that multiple copies of the trimer are attached and themultimerized envelope is prepared and formulated for immunization in ahuman. In certain embodiments, the compositions comprise envelopes,including but not limited to trimers as particulate, high-density arrayon liposomes or other particles, for example but not limited tonanoparticles. In some embodiments, the trimers are in a well ordered,near native like or closed conformation. In some embodiments the trimercompositions comprise a homogenous mix of native like trimers. In someembodiments the trimer compositions comprise at least 85%, 90%, 95%native like trimers.

The polypeptide contemplated by the invention can be a polypeptidecomprising any one of the polypeptides described herein. The polypeptidecontemplated by the invention can be a polypeptide consistingessentially of any one of the polypeptides described herein. Thepolypeptide contemplated by the invention can be a polypeptideconsisting of any one of the polypeptides described herein. In certainembodiments, the polypeptide is recombinantly produced. In certainembodiments, the polypeptides and nucleic acids of the invention aresuitable for use as an immunogen, for example to be administered in ahuman subject.

In certain embodiments the envelope is any of the forms of HIV-1envelope. In certain embodiments the envelope is gp120, gp140, gp145(i.e. with a transmembrane), gp150. In certain embodiments, gp140designed to form a stable trimer (See Tables 1A-B, FIGS. 22-24 , FIG. 59, FIGS. 80-82 , Example 9 for non-limiting examples of sequences ofstable trimer designs). In certain embodiments envelope protomers form atrimer which is not a SOSIP timer. In certain embodiment the trimer is aSOSIP based trimer wherein each protomer comprises additionalmodifications. In certain embodiments, envelope trimers arerecombinantly produced. In certain embodiments, envelope trimers arepurified from cellular recombinant fractions by antibody binding andreconstituted in lipid comprising formulations. See for exampleWO2015/127108 titled “Trimeric HIV-1 envelopes and uses thereof” whichcontent is herein incorporated by reference in its entirety. In certainembodiments the envelopes of the invention are engineered and comprisenon-naturally occurring modifications.

In certain embodiments, the envelope is in a liposome. In certainembodiments the envelope comprises a transmembrane domain with acytoplasmic tail embedded in a liposome. In certain embodiments, thenucleic acid comprises a nucleic acid sequence which encodes a gp120,gp140, gp145, gp150, gp160.

In certain embodiments, where the nucleic acids are operably linked to apromoter and inserted in a vector, the vectors is any suitable vector.Non-limiting examples, include, VSV, replicating rAdenovirus type 4,MVA, Chimp adenovirus vectors, pox vectors, and the like. In certainembodiments, the nucleic acids are administered in NanoTaxi blockpolymer nanospheres. In certain embodiments, the composition and methodscomprise an adjuvant. Non-limiting examples include, AS01 B, AS01 E,gla/SE, alum, Poly I poly C (poly IC), polylC/long chain (LC) TLRagonists, TLR7/8 and 9 agonists, or a combination of TLR7/8 and TLR9agonists (see Moody et al. (2014) J. Virol. March 2014 vol. 88 no. 63329-3339), or any other adjuvant. Non-limiting examples of TLR7/8agonist include TLR7/8 ligands, Gardiquimod, Imiquimod and R848(resiquimod). A non-limiting embodiment of a combination of TLR7/8 andTLR9 agonist comprises R848 and oCpG in STS (see Moody et al. (2014) J.Virol. March 2014 vol. 88 no. 6 3329-3339).

In certain aspects the invention provides a cell comprising a nucleicacid encoding any one of the envelopes of the invention suitable forrecombinant expression. In certain aspects, the invention provides aclonally derived population of cells encoding any one of the envelopesof the invention suitable for recombinant expression. In certainaspects, the invention provides a sable pool of cells encoding any oneof the envelopes of the invention suitable for recombinant expression.

In certain aspects, the invention provides a recombinant HIV-1 envelopepolypeptide from Tables 1A-B, wherein the polypeptide is a non-naturallyoccurring protomer designed to form an envelope trimer. The inventionalso provides nucleic acids encoding these recombinant polypeptides.Non-limiting examples of amino acids and nucleic acid of such protomersare shown in FIGS. 22-24 , FIG. 59 , FIGS. 80-82 .

In certain aspects the invention provides a recombinant trimercomprising three identical protomers of an envelope from Tables 1A-B. Incertain aspects the invention provides an immunogenic compositioncomprising the recombinant trimer and a carrier, wherein the trimercomprises three identical protomers of an HIV-1 envelope listed inTables 1A-B. In certain aspects the invention provides an immunogeniccomposition comprising nucleic acid encoding these recombinant HIV-1envelopes and a carrier.

In certain aspects the invention provides a selection of HIV-1 envelopesor any suitable form of a nucleic acid encoding HIV-1 envelope for usein an immunization regimen, wherein the selections of envelopescomprises envelopes M5, M11, w20.14, w30.20, w30.12, and w136.B18(Selection F, e.g. listed in FIG. 18A) or any combination thereof,envelopes M5, w30.25, w53.25, and w53.29 (Selection G, e.g. FIG. 20 ) orany combination thereof, envelopes M5, w30.20, w20.14, and w30.12(Selection H, e.g. FIG. 21 ) or any combination thereof. In certainaspects the invention provides a selection of HIV-1 envelopes forimmunization wherein the HIV-1 envelope is a loop D mutant envelope M5and/or M11. In certain embodiments the prime is M5.

In certain aspects the invention provides a selection of nucleic acidsencoding HIV-1 envelopes for immunization wherein the nucleic acidencodes a gp120 envelope, gp120D8 envelope, a gp140 envelope (gp140C,gp140CF, gp140CFI) as soluble or stabilized protomer of a SOSIP trimer,a gp145 envelope, a gp150 envelope, or a transmembrane bound envelope.

In certain aspects the invention provides a selection of HIV-1 envelopesfor immunization wherein the HIV-1 envelope is a gp120 envelope or agp120D8 variant. In certain embodiments a composition for immunizationcomprises protomers that form stabilized trimers, e.g. but not limitedto SOSIP.III trimers.

In certain embodiments, the compositions for use in immunization furthercomprise an adjuvant.

In certain embodiments, wherein the compositions comprise a nucleicacid, the nucleic acid is operably linked to a promoter, and could beinserted in an expression vector.

In certain aspects, the invention provides a kit comprising acombination/selection of immunogens of from Tables 1A-B, wherein thepolypeptide is a non-naturally occurring protomer designed to form anenvelope trimer. In certain aspects, the invention provides a kitcomprising a combination/selection of immunogens of from FIGS. 22-24 .In some embodiments the kit comprises instructions on how to carry outthe immunization regimen, including which immunogen(s) are a primeimmunization and which immunogen(s) comprise a boost immunization. Insome embodiments the kit comprises instructions on administration of theselection of immunogens as a prime or boost as part of a prime/boostimmunization regimen. In certain aspects, the invention provides a kitcomprising any one of the immunogens from Tables 1A-B, wherein thepolypeptide is a non-naturally occurring protomer designed to form anenvelope trimer and instructions on how to carry out an immunizationregimen with the immunogen of the kit. In some embodiments the kitcomprises instructions on administration of the immunogen as a prime oras a boost as part of a prime/boost immunization regimen. In someembodiments the immunogen could be administered sequentially oradditively. In certain aspects, the invention provides a kit comprisinga combination/selection of immunogens of from FIGS. 22-24 .

In one aspect the invention provides a composition for a prime boostimmunization regimen comprising one or more envelopes from Tables 1A-B,wherein the polypeptide is a non-naturally occurring protomer designedto form an envelope trimer, wherein the envelope is a prime or boostimmunogen. In one aspect the invention provides a composition for aprime boost immunization regimen comprising one or more envelopes fromFIGS. 22-24 wherein the envelope is a prime or boost immunogen.

In certain aspects the invention provides methods of inducing an immuneresponse in a subject comprising administering a composition comprisingany suitable form of a nucleic acid(s) encoding an HIV-1 envelope(s) inan amount sufficient to induce an immune response from one or more ofthe following groups: (a) the selection of envelopes M5, M11, w20.14,w30.20, w30.12, and w136.B18 (Selection F, e.g. listed in FIG. 18A) orany combination thereof; (b) envelopes M5, w30.25, w53.25, and w53.29(Selection G, e.g. FIG. 20 ) or any combination thereof; (c) envelopesM5, w30.20, w20.14, and w30.12 (Selection H, e.g. FIG. 21 ) or anycombination thereof and wherein the administration step canalternatively, or in addition, comprise administering an HIV-1polypeptide(s) in an amount sufficient to induce an immune response fromone or more of the following groups: (a) envelopes M5, M11, w20.14,w30.20, w30.12, and w136.B18 (Selection F, e.g. listed in FIG. 18A) orany combination thereof; (b) envelopes M5, w30.25, w53.25, and w53.29(Selection G, e.g. FIG. 20 ) or any combination thereof; (c) envelopesM5, w30.20, w20.14, and w30.12 (Selection H, e.g. FIG. 21 ) or anycombination thereof. In certain embodiments, the composition comprisesM5 or a nucleic acid encoding M5 that is administered as a primeimmunogen. In certain embodiments, the methods further compriseadministering M11 or a nucleic acid encoding M11. In certainembodiments, the methods further comprise administering HIV-1 envelopew20.14 or a nucleic acid encoding HIV-1 envelope w20.14, followed byadministering HIV-1 envelope w30.20 or a nucleic acid encoding HIV-1envelope w30.20, and followed by administering HIV-1 envelope w30.12 ora nucleic acid encoding HIV-1 envelope w30.12. In certain embodiments,the methods further comprise administering HIV-1 envelope w136.B18 or anucleic acid encoding HIV-1 envelope w136.B18.

In certain embodiments, the methods further comprise administering HIV-1envelope w30.25 or a nucleic acid encoding HIV-1 envelope w30.25, HIV-1envelope w53.25 or a nucleic acid encoding HIV-1 envelope w53.25, HIV-1envelope w53.29 or a nucleic acid encoding HIV-1 envelope w53.29.

In certain embodiments, the nucleic acid encodes a gp120 envelope,gp120D8 envelope, a gp140 envelope (gp140C, gp140CF, gp140CFI) assoluble or stabilized protomer of a SOSIP trimer, a gp145 envelope, agp150 envelope, or a transmembrane bound envelope. In certainembodiments, the polypeptide is gp120 envelope, gp120D8 envelope, agp140 envelope (gp140C, gp140CF, gp140CFI) as soluble or stabilizedprotomer of a SOSIP trimer, a gp145 envelope, a gp150 envelope, or atransmembrane bound envelope.

In certain aspects, the invention provides a method of inducing animmune response in a subject comprising administering a compositioncomprising envelope CH505 T/F, followed by envelope w53.16, followed byenvelope w78.33 and followed by envelope w100.B6, wherein eachcomposition comprises the envelope as a trimer. In certain embodimentsof the method the selection of immunogens is administered as nucleicacids.

In certain embodiments, the methods comprise administering an adjuvant.In certain embodiments, the methods comprise administering an agentwhich modulates host immune tolerance. In certain embodiments, theadministered polypeptide is multimerized in a liposome or nanoparticle.In certain embodiments, the methods comprise administering one or moreadditional HIV-1 immunogens to induce a T cell response. Non-limitingexamples include gag, nef, pol, etc.

In certain aspects, the invention provides a recombinant HIV-1 Envectodomain trimer, comprising three gp120-gp41 protomers comprising agp120 polypeptide and a gp41 ectodomain, wherein each protomer is thesame and each protomer comprises portions from envelope BG505 HIV-1strain and gp120 polypeptide portions from a CH505 HIV-1 strain andstabilizing mutations A316W and E64K, (see e.g. FIG. 23 ). In certainembodiments, the trimer is stabilized in a prefusion mature closedconformation, and wherein the trimer does not comprise non-naturaldisulfide bond between cysteine substitutions at positions 201 and 433of the HXB2 reference sequence. Non-limited examples of envelopescontemplated as trimers are listed in Tables 1A-B. In some embodiments,the amino acid sequence of one monomer comprised in the trimer is shownin FIG. 22-24 , FIG. 59 , FIGS. 80-82 . In some embodiments, the trimeris immunogenic. In some embodiments the trimer binds to any one of theantibodies PGT145, PGT151, CH103UCA, CH103, VRC01, PGT128, or anycombination thereof. In some embodiments the trimer does not bind toantibody 19B and/or 17B.

In certain aspects, the invention provides a pharmaceutical compositioncomprising any one of the recombinant trimers of the invention. Incertain embodiments the compositions comprising trimers are immunogenic.The percent trimer in such immunogenic compositions could vary. In someembodiments the composition comprises 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% stabilized trimer.

In certain aspects the invention provides any suitable form of a nucleicacid encoding a HIV-1 envelope from the selections of envelopes listedin FIG. 14A (envelopes M5, M11, 20.14, 30.28, 30.23. and 136.B18), FIG.15A (envelopes M5, M11, 20.14, 30.20, 30.23. and 136.B18), FIG. 16A(envelopes M5, M11, 20.14, 30.20, 30.12. and 136.B18), FIG. 18A(envelopes M5, M11, 20.14, 30.20, 30.12, and 136.B18), FIG. 20 (M5,30.25; 53.25; and 53.29), FIG. 21 (M5, w30.20, w20.14, w30.12), or anycombination thereof. In certain embodiments the envelopes bindpreferentially to an antibody or antibodies from CH103 lineage. Incertain embodiments the envelopes bind preferentially to an antibody orantibodies from CH235 lineage. In certain aspects the invention providesa polypeptide from the selections of envelopes listed in FIG. 14A(envelopes M5, M11, 20.14, 30.28, 30.23. and 136.B18), FIG. 15A(envelopes M5, M11, 20.14, 30.20, 30.23. and 136.B18), FIG. 16A(envelopes M5, M11, 20.14, 30.20, 30.12. and 136.B18), FIG. 18A(envelopes M5, M11, 20.14, 30.20, 30.12, and 136.B18), FIG. 20 (M5,30.25; 53.25; and 53.29), FIG. 21 (M5, w30.20, w20.14, w30.12), or anycombination thereof. In certain aspects the invention provides acomposition comprising any suitable form of the nucleic acids of theinvention. In certain aspects the invention provides a compositioncomprising any suitable polypeptide, wherein the polypeptide isengineered and recombinantly produced.

BRIEF DESCRIPTION OF THE DRAWINGS

To conform to the requirements for PCT patent applications, many of thefigures presented herein are black and white representations of imagesoriginally created in color.

FIG. 1 shows sequences of six envelopes (SEQ ID NOS 19-67, respectivelyin order of appearance): CH505.M5gp145, CH505.M11gp145,CH505w020.14gp145, CH505w030.28gp145, CH505w078.15gp145,CH505w053.31gp145, also as gp120D8 and gp160 amino acid and nucleic acidsequences. SEQ ID NOS 19-67 are included in the Sequence Listing whichis submitted electronically herewith in ASCII format and is herebyincorporated by reference in its entirety and forms part of thespecification.

FIG. 2A shows sequences of ten envelopes (SEQ ID NOS 68-98,respectively, in order of appearance): CH505.M5gp145, CH505.M11gp145,CH505w020.14gp145, CH505w030.28gp145, CH505w078.15gp145,CH505w53.16gp145, CH505w30.21gp145, CH505w78.33gp145, CH505w100.B6gp145,CH505w053.31gp145, amino acid and nucleic acid sequences. SEQ ID NOS68-98 are included in the Sequence Listing which is submittedelectronically herewith in ASCII format and is hereby incorporated byreference in its entirety and forms part of the specification.

FIG. 2B shows sequences of ten envelopes (SEQ ID NOS 99-124,respectively, in order of appearance): CH505.M5D8gp120,CH505.M11D8gp120, CH505w020.14D8gp120, CH505w030.28D8gp120,CH505w078.15D8gp120, CH505w053.16D8gp120, CH505w030.21D8gp120,CH505w078.33D8gp120, CH505w100.B6D8gp120, CH505w053.31D8gp120 as aminoacids and nucleic acids. SEQ ID NOS 99-124 are included in the SequenceListing which is submitted electronically herewith in ASCII format andis hereby incorporated by reference in its entirety and forms part ofthe specification.

FIG. 2C shows sequences of ten envelopes of FIG. 2B as gp160 amino acidand nucleic acid sequences (SEQ ID NOS 125-144, respectively, in orderof appearance). SEQ ID NOS 125-144 are included in the Sequence Listingwhich is submitted electronically herewith in ASCII format and is herebyincorporated by reference in its entirety and forms part of thespecification.

FIGS. 3A-C shows the genotype variation (A, left panel), neutralizationtiters (B, center panel), and Envelope phylogenetic relations (C, rightpanel) among CH505 Envelope variants. The vertical position in eachpanel corresponds to the same CH505 Env clone named on the right side ofthe tree. Distance from the Transmitted/Founder form generally increasesfrom top towards bottom of the figure. In the left panel (A), sites notcolored correspond to the Transmitted/Founder virus, red sites showmutations, and black sites correspond to insertions or deletionsrelative to the Transmitted/Founder virus. Additional annotationindicates the known CD4 binding-site contacts (short, vertical blackbars towards top), CH103 binding-site contacts for the resolvedstructure (short, vertical blue bars with a horizontal line to indicatethe region resolved by X-Ray Crystallography), gp120 landmarks (verticalgrey rectangular regions, V1-V5 hypervariable loops, Loop D, and CD4Loops), a dashed vertical line delineating the gp120/gp41 boundary, andresults from testing for CTL epitopes with ELISpot assays (magenta bandsat top and bottom show where peptides were tested and negative, and amagenta rectangle for the tested positive region outside the C-terminalend of V4). The center panel (B) depicts IC50 (50% inhibitoryconcentrations, in μg/ml) values from autologous neutralization assaysagainst 13 monoclonal antibodies (MAbs) of the CH103 lineage and each of134 CH505 Env-pseudotyped viruses. Color-scale values indicateneutralization potency and range from grey (no neutralization detected)through dark red (potent neutralization, i.e. <0.2 μg/ml; empty cellscorrespond to absence of information). The cumulative progression ofneutralization potency from left to right, corresponding todevelopmental stages in the CH103 lineage, indicates accumulation ofneutralization potency. Similarly, increased presence neutralizationsignal from top to bottom corresponds to increasing neutralizationbreadth per MAb in the CH103 lineage. In the right-most panel (C) is thephylogeny of CH505 Envs, with the x-axis indicating distance from theTransmitted-Founder virus per the scale bar (units are mutations persite). The tree is ordered vertically such that lineages with the mostdescendants appear towards the bottom. Each leaf on the tree correspondsto a CH505 autologous Env, with the name of the sequence depicted (‘w’and symbol color indicate the sample time-point; ‘M’ indicates asynthetic mutant Env). The color of text in each leaf name indicates itsinclusion in a possible embodiment, or grey for exclusion from anyembodiments described herein. Three long, vertical lines to the left ofthe tree depict the phylogenetic distribution of envelopes in threedistinct alternative embodiments (identified as “Vaccination Regimes1-3”), with diamonds used to identify each.

FIGS. 4-8 show Heat Map of Binding (log Area Under the Curve, AUC) ofSequential Envs to CH103 and CH235 CD4 Binding Site Broadly NeutralizingAntibody Lineages members. Numerical data corresponding to the graphicrepresentations in these figures are shown in Tables 2-5 in Example 2.

FIG. 9 shows neutralization activity of CH103 clonal lineage antibodiesagainst autologous CH505 viruses.

FIG. 10 shows neutralization susceptibility of the CH505 loop D mutantsto CH103 lineage antibodies.

FIG. 11 shows CH103 ELISA binding data and choice of immunogens.

FIG. 12 shows neutralization susceptibility of CH505 loop D mutants toCH235 lineage antibodies.

FIG. 13 shows neutralization activity of CH235 clonal lineage antibodiesagainst autologous CH505 viruses.

FIGS. 14A-B show a heat map of binding log Area Under the Curve, AUC) ofSequential Envs M5, M11, 20.14, 30.28, 30.23, 136.B18 to CH103 (FIG.14B) and CH235 (FIG. 14A-includes lineage member CH557) CD4 Binding SiteBroadly Neutralizing Antibody Lineages members.

FIGS. 15A-B show a heat map of binding log Area Under the Curve, AUC) ofSequential Envs M5, M11, 20.14, 30.20, 30.23, 136.B18 to CH103 (FIG.15B) and CH235 (FIG. 15A-includes lineage member CH557) CD4 Binding SiteBroadly Neutralizing Antibody Lineages members. Env 30.20 has betterprogression for CH235 whereas 30.28 has better progression for CH103,however early CH103 intermediates are covered well by 20.14.

FIGS. 16A-B show a binding log Area Under the Curve, AUC) of SequentialEnvs M5, M11, 20.14, 30.20, 30.12, 136.B18 to CH103 (FIG. 16B) and CH235(FIG. 16A-includes lineage member CH557) CD4 Binding Site BroadlyNeutralizing Antibody Lineages members.

FIGS. 17A-B show amino acid and nucleic acid sequences of M5, M11,20.14, 30.20, 30.12, 136.B18 envelopes: FIG. 17A shows sequences ofgp120D8 variants (SEQ ID NOS 145-156, respectively, in order ofappearance), FIG. 17B shows sequences of gp160 envelopes (SEQ ID NOS157-168, respectively, in order of appearance). SEQ ID NOS 145-168 areincluded in the Sequence Listing which is submitted electronicallyherewith in ASCII format and is hereby incorporated by reference in itsentirety and forms part of the specification.

FIGS. 18A-B (see also FIGS. 52A-B from Example 8) show. CH505 gp120 EnvQuasi-species Selected as Optimized Immunogens to Induce Both CH235 andCH103-like bnAbs, related to FIGS. 46A-B (Ex. 8). (A) Heatmap of thebinding data of selected CH235 and CH103 lineage members to the CH505Env glycoproteins selected to be used as immunogens. Individual Envclone names and weeks of isolation are shown on the left. (A) shows abinding log Area Under the Curve, AUC) of Sequential Envs M5, M11,20.14, 30.20, 30.12, 136.B18 to CH235 (left panel) and CH103 (rightpanel) CD4 Binding Site Broadly Neutralizing Antibody Lineages members.(B) Affinity of gHgL of 1B2530, 8ANC131, VRC01, VRC-PG04 and VRC-CH31 toa panel of 15 heterologous gp120 envelope glycoproteins.

FIGS. 19A-B show nucleic acid and amino acid sequences of M5, M11,20.14, 30.20, 30.12, 136.B18 envelopes (SEQ ID NOS 169-194,respectively, in order of appearance). The highlighted portions indicatenon-coding sequences—one stop codon at the end of each nucleotidesequences is not highlighted. SEQ ID NOS 169-194 are included in theSequence Listing which is submitted electronically herewith in ASCIIformat and is hereby incorporated by reference in its entirety and formspart of the specification.

FIG. 20 shows a selection for a Sequential Vaccine. Heat Map of Binding(log Area Under the Curve, AUC) of Sequential Envs to CH235 VH1-46 typeof CD4 mimic, CD4 Binding Site Broadly Neutralizing Antibody LineageMembers for sequential immunization. X axis shows CH235 antibody lineagemembers, from UCA to mature antibodies, from left to right.

FIG. 21 shows a selection for a Sequential Vaccine. Heat Map of Binding(log Area Under the Curve, AUC) of Sequential Envs to CH235 VH1-46 typeof CD4 mimic, CD4 Binding Site Broadly Neutralizing Antibody LineageMembers for sequential immunization.

FIG. 22A shows CH505 chimeric 6R.SOSIP.664 design. The gp120 of CH505(right) except the c-terminal 37 amino acids was transplanted into thewell-characterized A.BG505 6R.SOSIP.664 (left). The transplantationdesign takes advantage of the enhanced stability of the A.BG505 strain.The resultant chimeric molecule (center) has the CH505 gp120 (yellow)fused to the 37 c-terminal amino acids of A.BG505 (blue) and the A.BG505gp41 (magenta).

FIG. 22B shows nucleic acid sequences of various trimer designs of FIG.23A (SEQ ID NOS 195-233, respectively, in order of appearance). SEQ IDNOS 195-233 are included in the Sequence Listing which is submittedelectronically herewith in ASCII format and is hereby incorporated byreference in its entirety and forms part of the specification.

FIG. 23A shows amino acid sequences of various trimer designs (SEQ IDNOS 234-272, respectively, in order of appearance). In some embodimentsthe leader sequence for these proteins is MPMGSLQPLATLYLLGMLVASVLA (SEQID NO: 273). SEQ ID NOS 234-273 are included in the Sequence Listingwhich is submitted electronically herewith in ASCII format and is herebyincorporated by reference in its entirety and forms part of thespecification.

FIG. 23B shows annotated sequence of SOSIP.III design (SEQ ID NOS274-276, respectively, in order of appearance).

FIG. 24A shows amino acid and nucleic acid sequences of designsCH505TF.6R.SOSIP.664.v4.1_AMBRCTA and AMBRCTAG, and designsCH505M5chim.6R.SOSIP.664v4.1_AMBRCTA and AMBRCTAG (SEQ ID NOS 277-288,290, 289, 292, 291, 294, 293, 295, and 296, respectively, in order ofappearance). See also Example 9.

FIGS. 24B, C and D show sortase designs and nucleic acid and proteinsequences (SEQ ID NOS 297-308, respectively, in order of appearance).

FIG. 24E shows the PGT151 antibody staining of 293F cells transientlytransfected with AMBRCTA and AMBRCTAG constructs of FIG. 24A. Only theTM constructs show surface expression of Trimeric Envelope.

FIG. 24F shows the quantification of SOSIP trimer in the supernatant ofcells transfected with the constructs of FIG. 24A

FIG. 24G shows ferritin designs (SEQ ID NOS 309-313, respectively, inorder of appearance).

FIG. 24H shows antigenicity of M5 SOSIPv4.1 ferritin particle.

FIG. 24I shows comparison of binding of the M5 trimer alone versus theM5 trimer multimerized on the ferritin particle.

FIG. 24J shows negative stain EM of M5 trimers on the ferritin particle.The ring in the middle is ferritin and the trimer is the spikes comingoff of the ring.

FIG. 25 shows design of rhesus macaque immunogenicity study. Theimmunization schedule is shown in this figure. The study compared theimmunogenicity of the CD40 targeted Env to the wildtype Env in a rhesusmacaque immunogneicity study. The macaques were immunizedintramuscularly and electroporated twice with DNA encoding the CH505 T/Fgp145. After DNA priming the macaques were administered sequential CH505recombinant gp140C oligomers from the transmitted founder virus, andweeks 53, 78, and 100. Three macaques were immunized with the CH505 Envsconjugated to CD40 and 4 macaques were administered the CH505 Env asgp140C envelopes. We examined binding antibody titer by ELISA,neutralizing antibody titers by the TZM-bl assay, and profiled theantibody repertoire by monoclonal antibody isolation. The study analyzedthe immunogenicity of wildtype and CD40-targeted Env using antibodyELISA binding, TZM-bl neutralization assay, and will isolate monoclonalantibodies.

FIG. 26 shows plasma IgG responses to CH505 transmitted/founder gp140.This figure shows the binding titers over time with each symbolrepresenting an individual macaque and the red line and symbolindicating those animals that received the wildtype Env. The macaquesthat were immunized with the Env conjugated to anti-CD40 are shown inblue. The titers in both groups was comparable until week 18 which was 2weeks after the second protein boost. After that boost with the week 53Env the wildtype group tended to have higher binding antibody titers.

FIG. 27 shows CH505 gp140 vaccination induces plasma blocking of CD4binding. The figure shows whether CD4 binding site antibodies werepresent in the plasma using competition ELISAs for soluble CD4 (shown onthe left and a bnAb from the CH103 lineage called CH106 shown on theright. We examined the plasma blocking activity shown on the y-axis overtime and found that at week 18 the CD4 binding site response wasdramatically reduced to near background levels in the CD40 IgG4—Envgroup compared to the wildtype Env which showed 70 and 80% blocking ofsoluble CD4 and CH106 respectively.

FIG. 28 shows that plasma IgG exhibits CD4 binding site-directed bindingto a resurfaced gp120 core. This figure shows the ability of the plasmaIgG from all four animals to bind to RSC3 or its CD4 knock out mutant.Shown here is the difference in binding between the wildtype RSC3 andthe CD4 binding site mutant over time.

FIG. 29 shows CH505 gp140 vaccination elicits high titers of autologoustier 1 virus neutralization. This figure shows the neutralization titersfor each macaque represented as ID50 reciprocal dilutions against a tier1 virus called CH505 w4.3 isolated from the CH505 individual early ininfection. All 4 animals generated relatively high titers of tier 1neutralizing antibodies beginning with the protein boost. The titerswere increased with subsequent boosts with the sequential vaccine, andnotably these tier 1 neutralizing antibodies were typical for our CH505Env vaccinations that have been performed in macaques.

FIG. 30 shows CH505 gp140 sequential vaccination boosts autologous tier2 CH505 TF virus neutralization. This figure shows autologous tier 2neutralization by the plasma from each macaque. The ID50 titers areshown for each macaque and we observed 1 of 4 macaques generatedautologous tier 2 neutralizing antibodies in the plasma. Detectableneutralization first occurred after the CH505 week 53 Env protein boostand increased with each boost. This result was striking since thismacaque was the first vaccinated macaque where we observedneutralization of the CH505 TF virus.

FIG. 31 shows autologous neutralizing antibodies against all four tier 2viruses increased with sequential boosting. This figure shows theautologous tier 2 neutralization analysis to include the tier 2 CH505viruses that comprise the sequential vaccination regimen. The samemacaque 6207 was able to neutralize all four tier 2 CH505 viruses. Theneutralization was detectable against 3 of 4 of the CH505 viruses afteronly 2 protein boosts, and by three boosts all 4 viruses wereneutralized. We saw the neutralizing titers continued to increase witheach boost.

FIG. 32 shows NHP 6207 neutralizes heterologous tier 2 virusrepresentative global isolates. While autologous tier 2 neutralizationis difficult to elicit, heterologous tier 2 neutralization is even morerare to observe in vaccinated primates. To determine whether tier 2heterologous breadth was elicited in macaque 6207 plasma we testedneutralization against heterologous tier 2 viruses selected to representthe global circulating viruses. We examined neutralization of this 12virus panel by the plasma at week 30-post 4 sequential protein boostsand week 36 post 5 sequential protein boosts. After 4 protein boosts twoheterologous tier 2 viruses were neutralized. After the subsequent boost9/12 of the viruses were neutralized. Although the titers were low thisantibody response appears boostable and is currently the broadest tier 2neutralization known to be achieved in a vaccinated primate.

FIG. 33 shows B cell sorting for CD4 binding site differentialantibodies. Memory B cells from two macaques were sorted and comparedthe presence of RSC3-reactive B cells that did not bind the CD4 knockout mutant version of the protein called delta RSC3. A representativeFACS plot is shown on the left for the NHP 6207 who possessed SCL70reactive plasma IgG and developed broadly neutralizing antibodies in itsplasma. For comparison we sorted RSC3-reactive B cells from NHP 6436,which did not have broad neutralization in the plasma but was the othermacaque that tested positive for auto antibodies. The NHP 6207 had arelatively large percentage of RSC3 reactive B cells whereas NHP6436 hadvery few.

FIG. 34 shows macaque 6207 has broad plasma neutralization andantibodies against Scl70. This figure shows autoreactivity measured inthe Athena assay for each of the four macaques that received thewildtype CH505 gp140C envelopes in vaccination. Median fluorescenceintensity for binding to each autoantigen listed on the x-axis isdepicted in separate graphs for each macaque. The positivity thresholdfor the assay is marked by the dotted line. Interestingly, the macaquethat possessed broad neutralization possessed binding antibodies to theautoantigen SCL70, which is correlated of the autoimmune diseasescleroderma. The antibodies were present prior to vaccination indicatedby the binding in the grey bar. One other macaque tested positive forautoantibodies, but it bound to a different autoantigen.

FIG. 35 shows a heterologous panel heatmap of IC50s.

FIG. 36 shows the global panel, grouped by bNAb sensitivity (rightpanel). Env mutations over time in subject CH505, by week is shown inthe left panel.

FIG. 37 shows V5 selection yields population breadth.

FIG. 38 shows mutations and V5 length.

FIG. 39 shows a selection of four envelopes from CH505 and their bindingto CH235 lineage antibodies.

FIG. 40 shows a selection of CH505 immunogens to drive both CH103 andCH235 CD4 binding site types of broad neutralizing B cell lineages.

FIG. 41 shows a lot (165CGD) of DH235UCAtkLL_v3_4A/293i when it was runover SEC resin. The SEC chromatogram shows a main peak, and highmolecular forms. Another lot (48 EML) of DH235UCAtkLL_v3_4A/293i has asimilar profile.

FIG. 42 shows the SEC profile of the main peak/fraction ofDH235UCAtkLL_v3_4A/293i (lot 170712PPF). Antibody purified over SECresin is described in Example 13. In some instances, this SEC antibodyis referred as a purified antibody.

FIG. 43 shows comparison of neutralization (IC₅₀ values) of two lots ofDH235UCAtkLL. Lot 48EML was not purified by SEC, and lot 170712PPF waspurified by SEC (See FIGS. 41 and 42 ). The identity of the neutralizedvirus (envelope) is listed in the first column. The first column alsoindicates whether the virus was grown in 293T cell or in GnTI−/− cells.In this figures, and throughout other figures, CH0505TF.M5 refers toCH505M5 sequence. CH505 M5 has the CH505 T/F sequence with a N279K aminoacid change.

FIG. 44 shows comparison neutralization (IC₅₀ values) of two lots ofDH235UCAtkLL for neutralization of various CH505 viruses. Viruses arelisted in the x-axis—N279X refers to various amino acids changes atposition 279. Lot 48EML was not purified by SEC, and lot 170712PPF waspurified by SEC (See FIGS. 41 and 42 ).

FIG. 45A shows neutralization results with SEC purifiedDH235UCAtkLL_v3_4A. Viruses and IC₅₀ values are listed in FIG. 45B.CH0505TF.M5 refers to CH505M5.

FIG. 46 shows binding of CH235UCA to various CH505 SOSIPs. The envelopemarked with * was also used in cryoEM studies. This figure shows thatneutralization Predicts nM Affinity Binding to CH0505 SOSIP.

FIG. 47 shows that DH235UCAtkLL Fab potency is remarkably weak comparedto IgG (>2 log reduction in IC50).

FIG. 48 shows the profile of intermediate antibodyDH235VH_I1_v2_4A/293i, lot 218SJA without SEC purification.

FIG. 49 shows the profile of intermediate antibody DH235_I1_v2_4A/293i,Lot 171017PPF after SEC purification.

FIG. 50 shows the profile of intermediate antibody DH235_I3_v2_4A/293i,lot 330JAH without SEC purification.

FIG. 51 shows the profile if intermediate antibody DH235_I3_v2_4A/293i,Lot 171013PPF after SEC purification.

FIG. 52 shows the profile of intermediate antibody DH235_I4_v2_4A/293i,lot 4RKK without SEC purification.

FIG. 53 shows the profile of intermediate antibody DH235_I4_v2_4A/293i,Lot 171018PPF after SEC purification.

FIG. 54 shows Cryo-EM Structure of CH235 UCA bound to HIV-1 EnvCH505M5chim.6R.SOSIP.664v4.1_G458Y/GnTi− at 5.4 Å resolution. The 3Dclass averages show 3 Fa bound trimer. These classes have structuraldifference and could not be aligned together. At the observedresolution, most side chains and glycans could not be visualized.

FIG. 55 shows a detail of interaction between CH235 UCA and envelope. Inthis model. Y458 interacts with W50 (CDR H2) and W94 (CDR L3). Otherbulky and hydrophobic residues (or Arg, which can form a pi-cationinteraction with the two Trps) at position 458 are expected to stabilizethis interaction as well.

FIG. 56 shows Derivation of Man5-Enriched Glycans on HIV-1 Env.

FIG. 57 shows summary of data suggesting that VRC01 resistance mutationsare potential germline targeting mutations for CH235.12 lineage.

FIGS. 58A and 58B show some embodiments of an immunization strategies toelicit CH235.12-like BNAbs. CH505 T/F N279K has the sequence and is alsoreferred to as CH505 M5 envelope. In certain embodiments, the immunogenslisted under prime are administered individually. In certainembodiments, the immunogens listed under prime are administeredsequentially as any combination of two immunogens or the combination ofthree. In certain embodiments, the immunogens listed under prime areadministered sequentially starting with the immunogen with lowestaffinity (e.g. CH505 T/F N279K G458Y grown in 293T cells). In certainembodiments, the immunogens under prime are administered sequentiallystarting with the immunogen with highest BCR affinity (e.g. CH505 T/FN279K G458Y grown in GnTI−/− cells). In certain embodiments, theimmunogens under “boost” are administered as follows: CH0505TF G458YN279K grown in 293T cells followed by CH505 T/F grown in 293T cells. Incertain embodiments, the immunogens under “boost” are administered asfollows: CH0505TF GnTI−/− followed by CH505 T/F grown in 293T cells.FIG. 58B shows Additional boosting immunogens could be used to increasematuration of antibodies. Other cells may be used in place of 293T cellsto produce naturally or fully glycosylated immunogens.

FIG. 59A shows amino acid and nucleic acid (FIG. 59B) sequence of VH andVL CH235 UCAs. FIG. 59A discloses SEQ ID NOS 397-400, respectively, inorder of appearance. FIG. 59B discloses SEQ ID NOS 401-404,respectively, in order of appearance. FIG. 59C shows amino and FIG. 59Dnucleic acid sequence of envelopes. FIG. 59C discloses SEQ ID NOS405-412, respectively, in order of appearance. FIG. 59D discloses SEQ IDNOS 413-420, respectively, in order of appearance. FIG. 59E an alignmentof CH505M5chim.6R.SOSIP.664v4.1 G458Y and CH505M5chim.6R.SOSIP.664v4.1.FIG. 59E discloses SEQ ID NOS 405 and 421, respectively, in ordrer ofappearance. FIG. 59F shows CH505 M5 gp120 produced at the DHVI GMPFacility binds to the CD4 binding site CH235 UCA with a Kd of 4,566 nMwhile the mature bnAb CH235 binds with at Kd of 8.0 nM.

FIG. 60A shows a model of M5 SOSIP Ferritin particle with 6 Env trimersdisplayed, based on ferritin and SOSIP trimer crystal structures. FIG.60B shows negative stained EMs of M5 SOSIP Ferritin particles purifiedby size exclusion chromatography. The number of particles vary becauseof the variability of orientation of the particles on the EM grid.

FIG. 61 shows stabilization of chimeric CH505 TF SOSIP gp140. Theintroduction of a cysteine at positions 201 and 433 formed a disulfidebond that stabilized the trimer in the pre-CD4 bound conformation (NatStruct Mol Biol. 2015 July; 22(7): 522-531). This mutation was alsoadded to further stabilize the CH505 chimeric SOSIP.

FIG. 62 shows CH505 SOSIP.I binds to trimer-specific bnAbs. The chimericCH505 TF SOSIP.I was produced and tested for binding to trimer specificbnAbs. In SPR assays, CH505 bound both PGT145 and PGT151.

FIG. 63 shows SOSIP.I—stabilization of the trimer to reduce CD4 bindingalso disrupts binding by the CH103 lineage. When the UCA of the CH103lineage or a mature bnAb from the lineage CH106 was assessed for bindingto the CH505 TF SOSIP.I neither antibody bound to the trimer. Incontrast the CD4 mimicking antibody VRC01 was still able to bind.

FIG. 64 shows CH103 UCA binds the CH505 transmitted founder gp120. Themonomeric CH505 TF gp120 binds to the CH103 UCA by SPR as shown in thebox.

FIG. 65 shows CH505 TF SOSIP.II—removal of the DS mutations to improveCD4bs Ab binding. To test whether the DS stabilizing mutations disruptedCH103 UCA binding, since they were reported to decrease CD4 binding, thecysteine mutations were reverted back to the alanine and isoleucinepresent in the wildtype virus. The antigenicity of these trimers, calledSOSIP.II, was tested.

FIG. 66 shows removal of the disulfide stabilizing bond improves UCAbinding. A summary of the binding of first SOSIP design called SOSIP.Ifor comparison to the SOSIP.II proteins is shown. The binding is heatmapped where the darker the color the stronger the binding in BLIexperiments. Identical to the first SOSIP design, PGT151 and PGT145bound to the SOSIP.II design relatively strongly indicating trimerformation. The CH103 lineage antibodies were also able to in bind theSOSIP.II version of the chimeric CH505 trimer. This design still had theV3 loop exposed and at least a portion of the trimers were in a CD4bound conformation as indicated by 19B and 17B binding.

FIG. 67 shows CH505 TF chimeric SOSIP.III—introduction of twostabilizing mutation to reduce V3 exposure. Two mutations that reducedV3 exposure in nonchimeric SOSIPs were tested to see if these mutationscould function similarly in the chimeric SOSIP design.

FIG. 68 shows CH505 TF chimeric SOSIP.III forms trimeric envelope andbinds the CH103 UCA. The two mutations were introduced and the abilityof this protein to form trimers was assessed by PGT151 binding and bynegative stain EM. As shown on the left this protein bound to the trimerspecific bnAb PGT151, and the protein formed a trimer as shown in thenegative stain EM on the right.

FIG. 69 shows CH103 UCA binds to the CH505 TF SOSIP.III. When binding ofthe SOSIP.III was assessed and compared to the CH505 TF gp120, it wasobserved that the off-rate for the CH103 UCA was 10-fold better for thetrimer which resulted in an approved affinity for the timer compared tothe gp120.

FIG. 70 shows affinity maturation to the TF SOSIPv4.1 correlates withneutralization potency. The binding of the members of the CH103 lineageto the CCH505 TF was assessed. A positive correlation between affinityfor the CH505 TF SOSIP.III was found, shown here on the y-axis andbottom row of the table and neutralization potency against the CH505 TFvirus shown here on the x-axis and in the table. As antibodies affinitymatured and could bind more strongly to the CH505 TF trimer, they alsowere able to neutralize the CH505 virus more potently.

FIG. 71 shows the CH103 mature bnAb engages each protomer of the CH505TF SOSIP.III. The high affinity of the CH103 mature antibodies for thesetrimers provided an opportunity to study the stoichiometry of theinteraction of the bnAb CH103 and its autologous Envelope. Theunliganded trimer formed trimers and upon incubating it with CH103 Fabthree CH103 Fabs were observed indicated by the arrows bound to eachtrimer. Thus the CH103 and gp140 had a 1 to 1 ratio for binding.

FIG. 72 shows rational design to improve the antigenicity of CH505 TFtrimers. The SOSIP.III had the desired antigenicity for the CH103lineage of antibodies. Its antigenicity on a larger panel of bnAbs wasassessed. It bound to bnAbs but unlike the SOSIP.II it did not bind to19B or 17B.

FIG. 73 shows CH505 Envs from sequential viruses form stable trimers aschimeric SOSIP.II and III.

This approach has been employed to multiple CH505 Env sequences in orderto make sequential vaccination regimens. A 4-valent vaccination regimenof SOSIP.II was made. The same 4—valent vaccine was made using theSOSIP.III design. A 6-valent vaccine can be made. Trimers to wereanalyzed for glycosylation and disulfide bond analysis and the Envs havethe expected glycosylation and lack aberrant disulfide bonds.

FIG. 74 shows the chimeric SOSIP.III design is applicable to diverseviruses. This design can be extrapolated to Envs that are not from theCH505 infected individual. Envs from clade C or AE or a group Mconsensus have all been used and form stable trimers using this design.This highlights the general applicability of this trimer design.

FIG. 75 shows increased potency of CD4bs bNAbs against Man5-enriched(GnTI^(−/)) HIV-1.

FIG. 76 shows CD4bs bNAbs that are more potent against targetedglycan-deleted, Man5-enriched viruses. SM=N276D; DM=N460D.N463D;TM=N276D.N460D.N463D.

FIG. 77 shows Detection of neutralizing activity by near-germline andintermediate forms of CD4bs bNAbs. Left: Germline-reverted VRC01 assayedagainst 426c, 426c single mutant (SM, N276D), 426c double mutant (DM,N460D/N463D) and 426c triple mutant (TM, N276D/N460D/N463D) produced ineither 293T cells or 293s/GnTI−/− cells. The germline-reverted VRC01contains mature CDRH3 and J regions whose germlines cannot be inferredwith existing sequence information. Right: Germline-reverted (UCA,unmutated common ancestor), four late intermediates (I4 is least mature,I1 is most mature) and mature VRC-CH31 assayed against 426c and 426c.DMproduced in 293T cells, and against 426c.DM produced in 293s/GnTI−/−cells (Man5-enriched). Man5-enriched versions of 426c.SM and 426c.TMwere not assayed because they lack a glycan at position 276 that thisantibody requires for optimal neutralization.

FIG. 78A-78B shows Detection and mapping of near germline-revertedvariants of VRC01-class bNAbs in the context of targeted glycan-deletedGnTI−/− virus. A. Neutralizing activity of the mature (black bars) andnear-germline forms (grey bars) of the indicated bNAbs. Positiveneutralization by near germline forms of bNAbs are indicated by anasterisk. B. Neutralizing activity of near germline forms of VRC01 classbNAbs against 426c.TM/GnTI−/− (black bars) and 426c.TM.D279K/GnTI−/−(grey bars). Horizontal dashed lines indicate the highest concentrationsof antibodies tested (25 μg/ml in A, 50 μg/ml in B).

FIG. 79A-79C shows Neutralization of CH0505.G458Y by three UCAs of CH235is seen when the virus is produced in 293s.GnTI−/− cells but not whenproduced in 293T cells. Data are summarized in Example 10 Table 3.

FIG. 80A-80C show structure and models of structure of CH235 antibodiesand gp120 envelopes. FIG. 80A shows Crystal Structure of gp120 and CH235(Ex. 8, published in Cell. 2016 Apr. 7; 165(2):449-63, PDB:5F9W). FIG.80B shows a model of CH235 UCA antibody interaction with gp120. FIG. 80Cshows a model of CH235 UCA antibody interaction with gp120 G458Ymutation. The figure shows a model of how G458Y provides improvedcontacts with ISO in CDRH2 of CH235 UCA heavy chain.

FIG. 81 shows amino acid sequences of envelopes with G458 mutation to Y(G458Y). FIG. 81 discloses SEQ ID NOS 422-461, 439, 438, 437, 441, 440,462, 451, 450, 449, 448, 447, 463, 459, 458, 457, 461, 460, 464, 431,430, 429, 428, 427, 465, 426, 425, 424, 423, 422, 466, 434, 433, 432,436, 435, 467, 444, 443, 442, 446, 445, 468, 455, 456, 454, 453, 452 and469, respectively, in order of appearance.

FIG. 82 shows one embodiment of nucleic acid sequences encoding gp160envelopes of FIG. 7 . FIG. 82 discloses SEQ ID NOS 470-477,respectively, in order of appearance.

FIG. 83A-B shows the increased potency of CD4bs bNAbs againstMan₅-enriched (GnTI⁻) HIV-1. (A) Env-pseudotyped viruses CE1176 and WITOwere produced in 293T cells (black bars) and 293S GnTI⁻ cells (greybars) and assessed for sensitivity to neutralization by three matureCD4bs bNAbs (VRC01, 3BNC117 and VRC-CH31) in TZM-bl cells. (B)Env-pseudotyped virus TRO.11 was produced in 293T and 293S GnTI⁻ cellsand assessed for sensitivity to neutralization in TZM-bl cells by apanel of mature bNAbs to multiple epitopes. Additional assays wereperformed with germline-reverted forms of CD4bs bNAbs. Black asterisksindicate CD4bs bNAb that were more potent against virus produced in 293SGnTI⁻ cells. A red asterisk highlights a case where neutralization wasless potent against the 293S GnTI⁻ virus.

FIG. 84A-84B shows the complementarity of targeted glycan-deletion andMan₅-enrichement for neutralization by germline-reverted VRC01. (A)Parental and glycan deletion mutants of 426c were produced asEnv-pseudotyped viruses in 293T and 293S GnTI⁻ cells and assayed forneutralization by five mature CD4bs bNAbs in TZM-bl cells. The 426cmutants were SM (N276D), DM (N460D.N463D), TM (N276D.N460D.N463D) andTM4 (S278R.G471S.N460D.N463D). Horizontal dotted lines indicate thehighest concentration of bNAb tested. (B) Germline-reverted VRC01 wasassayed against 426c, 426c.SM, 426c.DM, 426c.TM and 426c.TM4 Envsproduced in 293T cells or 293S GnTI⁻ cells. Neutralization was dependenton both Man₅-enrichment and targeted glycan deletion. Thisgermline-reverted VRC01 contains mature CDRH3 and J regions whosegermlines cannot be inferred with existing sequence information.

FIG. 85A-85B shows the detection and epitope mapping of neutralizationby germline forms of VRC01-class bNAbs. (A) Germline reverted forms ofthe indicated bNAbs were assayed in TZM-bl cells against 426c.TM and426c.TM4 Env-pseudotyped viruses produced in 293S GnTI⁻ cells. Thedotted line indicates the highest concentration of antibody tested (25μg/ml). (B) Germline forms of VRC01, VRC07 and VRC20 were assayed inTZM-bl cells against Env 426c.TM (black bars) and Env 426c.TM.D279K(grey bars) produced in 293S GnTI⁻ cells. The dotted line indicates thehighest concentration of antibody tested (50 μg/ml).

FIG. 86 shows the neutralization by intermediates of VRC-CH31. InferredUCA, four late intermediates (I4 is least mature, I1 is most mature) andmature VRC-CH31 were assayed against 426c and 426c.DM Envs produced in293T cells, and against Env 426c.DM produced in 293S GnTI⁻ cells. GnTI⁻versions of 426c.SM and 426c.TM Envs were not assayed because they lackthe N276 glycan that VRC01-CH31 requires. A horizontal dashed line isused to show the highest concentration tested (40 μg/ml for CH103, 25μg/ml for VRC-CH31).

FIG. 87A-B shows the neutralization by intermediates of CH103 and CH235in the context of targeted glycan-deleted autologous Envs produced in293S GnTI⁻ cells. Targeted glycan deleted variants of Env CH0505TF wereproduced in either 293T cells (back bars) or 293S GnTI⁻ cells (greybars) and assayed in TZM-bl cells. Assays were performed with UCAs,intermediates and mature forms of CH103 (A) and CH235 (B). Thehorizontal lines indicate the highest concentration of antibody tested(50 μg/ml).

FIG. 88A-C shows the neutralization by germline-reverted CH235 requiresboth Man₅ enrichment and mutation of G458 in gp120. (A) CH235 UCA2 wasassayed for neutralizing activity against CH0505TF that was produced inGnTI⁻ cells and contained different amino acid substitutions at position458 of gp120. (B) Hydrophobicity of the amino acid substitutions atposition 458 is correlated with CH235 UCA2 neutralization potency(Pearson's r=0.78). The hydrophobicity scale is oriented such thatnegative values correspond to more hydrophobic residues. The log IC50scale is oriented such that negative values correspond to greaterneutralization potency (neutralization achieved at lower antibodyconcentrations). (C) G458Y provides improved contacts with 150 in CDRH2of CH235 UCA heavy chain. In these structures, the CD4-binding site ongp120 is shown as green space filled structure and the CDRH2 of CH235 isshown as blue ribbon structure. From the crystal structure of thewild-type gp120-CH235 complex (left panel), G458 (shown in magenta)) issmall, and makes contact with the large aromatic rings of tryptophan(W50) of the DH235 CDRH2. In the DH235 UCA2, the residue at position 50is isoleucine (I50), a much smaller amino acid. Structural modelingrevealed that I50 does not reach into the cavity toward G458 (middlepanel). When G458 is mutated to the larger tyrosine (Y458), structuralmodeling of this mutation showed the aromatic ring from gp120 can reachinto the cavity to interact with the small isoleucine in CH235 UCA2(right panel).

FIG. 89 shows impact of amino acids other than tyrosine (Y) at Envposition 458.

FIG. 90 shows impact of amino acids other than lysine (K) at Envposition 279.

DETAILED DESCRIPTION OF THE INVENTION

The development of a safe, highly efficacious prophylactic HIV-1 vaccineis of paramount importance for the control and prevention of HIV-1infection. A major goal of HIV-1 vaccine development is the induction ofbroadly neutralizing antibodies (bnAbs) (Immunol. Rev. 254: 225-244,2013). BnAbs are protective in rhesus macaques against SHIV challenge,but as yet, are not induced by current vaccines.

The ability to stimulate germline B cells that give rise to broadlyneutralizing antibodies (bNAbs) is a major goal for HIV-1 vaccinedevelopment. bNAbs that target the CD4-binding site (CD4bs) and exhibitextraordinary potency and breadth of neutralization are particularlyattractive to elicit with vaccines. Glycans that border the CD4bs andimpede the binding of germline-reverted forms of CD4bs bNAbs arepotential barriers to naïve B-cell receptor engagement. In some aspects,pseudovirus neutralization was used as a means to identify Envmodifications that permit native Env trimer binding to germline revertedCD4bs bNAb CH235.12 (VH1-46). Two mutations (N279K.G458Y), when combinedwith Man5-enrichment of N-linked glycans that are otherwise processedinto complex glycans, rendered autologous CH0505TF Env highly sensitiveto neutralization by CH235.12 UCA. These findings suggest a vaccinestrategy to initiate and mature the CH235.12 lineage.

In some embodiments, site-directed mutagenesis was used to createmutants of autologous CH0505TF Env. Mutants were produced in 293T/17 and293S/GnTI-cells lacking the enzyme N-acetylglucosaminyltransferase(GnTI-) to enrich for Man5 glycoforms. Naturally-glycosylated andMan5-enriched forms of parental and mutant Envs were tested forneutralization by the unmutated common ancestor (UCA), intermediates,and mature forms of CH235.12. Various trimers comprising these mutationswere tested for UCA binding.

In some aspects, the paradigm of B cell lineage immunogen design (NatureBiotech. 30: 423, 2012) in which the induction of bnAb lineages isrecreated is also used to identify other immunogens for use in themethods of the invention. It was recently demonstrated the power ofmapping the co-evolution of bnAbs and founder virus for elucidating theEnv evolution pathways that lead to bnAb induction (Nature 496: 469,2013). From this type of work has come the hypothesis that bnAbinduction will require a selection of antigens to recreate the “swarms”of sequentially evolved viruses that occur in the setting of bnAbgeneration in vivo in HIV infection (Nature 496: 469, 2013).

A critical question is why the CH505 immunogens are better than otherimmunogens. This rationale comes from three recent observations. First,a series of immunizations of single putatively “optimized” or “native”trimers when used as an immunogen have not induced bnAbs as singleimmunogens. Second, in all the chronically infected individuals who dodevelop bnAbs, they develop them in plasma after ˜2 years. When theseindividuals have been studied at the time soon after transmission, theydo not make bnAbs immediately. Third, now that individual's virus andbnAb co-evolution has been mapped from the time of transmission to thedevelopment of bnAbs, the identification of the specific Envs that leadto bnAb development have been identified-thus taking the guess work outof envelope choice.

Two other considerations are important. The first is that for the CH103bnAb CD4 binding site lineage, the VH4-59 and Vλ3-1 genes are common asare the VDJ, VJ recombinations of the lineage (Liao, Nature 496: 469,2013). In addition, the bnAb sites are so unusual, we are finding thatthe same VH and VL usage is recurring in multiple individuals. Thus, wecan expect the CH505 Envs to induce CD4 binding site antibodies in manydifferent individuals.

Regarding the choice of gp120 vs. gp160, for the genetic immunization wewould normally not even consider not using gp160. However, in acuteinfection, gp41 non-neutralizing antibodies are dominant and overwhelmgp120 responses (Tomaras, G et al. J. Virol. 82: 12449, 2008; Liao, H Xet al. JEM 208: 2237, 2011). Recently we have found that the HVTN 505DNA prime, rAd5 vaccine trial that utilized gp140 as an immunogen, alsohad the dominant response of non-neutralizing gp41 antibodies. Thus, wewill evaluate early on the use of gp160 vs gp120 for gp41 dominance.

In certain aspects the invention provides a strategy for induction ofbnAbs is to select and develop immunogens and combinations designed torecreate the antigenic evolution of Envs that occur when bnAbs dodevelop in the context of infection.

That broadly neutralizing antibodies (bnAbs) occur in nearly all serafrom chronically infected HIV-1 subjects suggests anyone can developsome bnAb response if exposed to immunogens via vaccination. Workingback from mature bnAbs through intermediates enabled understanding theirdevelopment from the unmutated ancestor, and showed that antigenicdiversity preceded the development of population breadth. See Liao etal. (2013) Nature 496, 469-476. In this study, an individual “CH505” wasfollowed from HIV-1 transmission to development of broadly neutralizingantibodies. This individual developed antibodies targeted to CD4 bindingsite on gp120. In this individual the virus was sequenced over time, andbroadly neutralizing antibody clonal lineage (“CH103”) was isolated byantigen-specific B cell sorts, memory B cell culture, and amplified byVH/VL next generation pyrosequencing. The CH103 lineage began by bindingthe T/F virus, autologous neutralization evolved through somaticmutation and affinity maturation, escape from neutralization drove rapid(clearly by 20 weeks) accumulation of variation in the epitope, antibodybreadth followed this viral diversification.

Further analysis of envelopes and antibodies from the CH505 individualindicated that a non-CH103 Lineage (DH235=CH235) participates in drivingCH103-BnAb induction. See Gao et al. (2014) Cell 158:481-491. Forexample, V1 loop, V5 loop and CD4 binding site loop mutations escapefrom CH103 and are driven by CH103 lineage. Loop D mutations enhancedneutralization by CH103 lineage and are driven by another lineage.Transmitted/founder Env, or another early envelope for example W004.26,triggers naïve B cell with CH103 Unmutated Common Ancestor (UCA) whichdevelop in to intermediate antibodies. Transmitted/founder Env, oranother early envelope for example W004.26, also triggers non-CH103autologous neutralizing Abs that drive loop D mutations in Env that haveenhanced binding to intermediate and mature CH103 antibodies and driveremainder of the lineage. In certain embodiments, the inventivecomposition and methods also comprise loop D mutant envelopes (e.g. butnot limited to M10, M11, M19, M20, M21, M5, M6, M7, M8, M9) asimmunogens. In certain embodiments, the D-loop mutants are included inan inventive composition used to induce an immune response in a subject.In certain embodiments, the D-loop mutants are included in a compositionused as a prime.

The invention provides various methods to choose a subset of viralvariants, including but not limited to envelopes, to investigate therole of antigenic diversity in serial samples. In other aspects, theinvention provides compositions comprising viral variants, for examplebut not limited to envelopes, selected based on various criteria asdescribed herein to be used as immunogens. In some embodiments, theimmunogens are selected based on the envelope binding to the UCA, and/orintermediate antibodies. In some embodiments the immunogens are selectedbased on their chronological appearance and/or sequence diversity duringinfection.

In other aspects, the invention provides immunization strategies usingthe selections of immunogens to induce cross-reactive neutralizingantibodies. In certain aspects, the immunization strategies as describedherein are referred to as “swarm” immunizations to reflect that multipleenvelopes are used to induce immune responses. The multiple envelopes ina swarm could be combined in various immunization protocols of primingand boosting. Immune responses, including B cell and T cell responses,could be measured by any suitable assay and criteria, such as but nonlimited plasma neutralization, plasma binding to vaccine and/orheterologous envelopes and/or viruses could be measured.

In certain embodiments the invention provides that sites losing theancestral, transmitted-founder (T/F) state are most likely underpositive selection. From acute, homogenous infections with 3-5 years offollow-up, identified herein are sites of interest among plasma singlegenome analysis (SGA) Envs by comparing the proportion of sequences pertime-point in the T/F state with a threshold, typically 5%. Sites withT/F frequencies below threshold are putative escapes. We then selectedclones with representative escape mutations. Where more information wasavailable, such as tree-corrected neutralization signatures and antibodycontacts from co-crystal structure, additional sites of interest wereconsidered.

Co-evolution of a broadly neutralizing HIV-1 antibody (CH103) andfounder virus was previously reported in African donor (CH505). See Liaoet al. (2013) Nature 496, 469-476. In CH505, which had an early antibodythat bound autologous T/F virus, we studied 398 envs from 14 time-pointsover three years (median per sample: 25, range: 18-53). We found 36sites with T/F frequencies under 20% in any sample. Neutralization andstructure data identified 28 and 22 interesting sites, respectively.Together, six gp41 and 53 gp120 sites were identified, plus six V1 or V5insertions not in HXB2.

The invention provides an approach to select reagents for neutralizationassays and subsequently investigate affinity maturation, autologousneutralization, and the transition to heterologous neutralization andbreadth. Given the sustained coevolution of immunity and escape thisantigen selection based on antibody and antigen coevolution has specificimplications for selection of immunogens for vaccine design.

In one embodiment, five envelopes were selected that represent envelopeantigenic diversity. In another embodiment, six envelopes were selectedthat represent envelope antigenic diversity. In another embodiment, tenenvelopes were selected that represent envelope antigenic diversity.These sets of envelopes represent antigenic diversity by deliberateinclusion of polymorphisms that result from immune selection byneutralizing antibodies. These selections represent various levels ofantigenic diversity in the HIV-1 envelope. In some embodiments theselections are based on the genetic diversity of longitudinally sampledSGA envelopes. In some embodiments the selections are based on antigenicand or neutralization diversity. In some embodiments the selections arebased on the genetic diversity of longitudinally sampled SGA envelopes,and correlated with other factors such as antigenic/neutralizationdiversity, and antibody coevolution.

Sequences/Clones

Described herein are nucleic and amino acids sequences of HIV-1envelopes. The sequences for use as immunogens are in any suitable form.In certain embodiments, the described HIV-1 envelope sequences aregp160s. In certain embodiments, the described HIV-1 envelope sequencesare gp120s. Other sequences, for example but not limited to stable SOSIPtrimer designs, gp145s, gp140s, both cleaved and uncleaved, gp140 Envswith the deletion of the cleavage (C) site, fusion (F) andimmunodominant (I) region in gp41—named as gp140ΔCFI (gp140CFI), gp140Envs with the deletion of only the cleavage (C) site and fusion (F)domain—named as gp140ΔCF (gp140CF), gp140 Envs with the deletion of onlythe cleavage (C)—named gp140ΔC (gp140C) (See e.g. Liao et al. Virology2006, 353, 268-282), gp150s, gp41s, which are readily derived from thenucleic acid and amino acid gp160 sequences. In certain embodiments thenucleic acid sequences are codon optimized for optimal expression in ahost cell, for example a mammalian cell, a rBCG cell or any othersuitable expression system.

An HIV-1 envelope has various structurally defined fragments/forms:gp160; gp140—including cleaved gp140 and uncleaved gp140 (gp140C),gp140CF, or gp140CFL gp120 and gp41. A skilled artisan appreciates thatthese fragments/forms are defined not necessarily by their crystalstructure, but by their design and bounds within the full length of thegp160 envelope. While the specific consecutive amino acid sequences ofenvelopes from different strains are different, the bounds and design ofthese forms are well known and characterized in the art.

For example, it is well known in the art that during its transport tothe cell surface, the gp160 polypeptide is processed and proteolyticallycleaved to gp120 and gp41 proteins. Cleavages of gp160 to gp120 and gp41occurs at a conserved cleavage site “REKR.” (SEQ ID NO: 1) SeeChakrabarti et al. Journal of Virology vol. 76, pp. 5357-5368 (2002) seefor example FIG. 1 , and Second paragraph in the Introduction on p.5357; Binley et al. Journal of Virology vol. 76, pp. 2606-2616 (2002)for example at Abstract; Gao et al. Journal of Virology vol. 79, pp.1154-1163 (2005); Liao et al. Virology vol. 353(2): 268-282 (2006).

The role of the furin cleavage site was well understood both in terms ofimproving cleave efficiency, see Binley et al. supra, and eliminatingcleavage, see Bosch and Pawlita, Virology 64 (5):2337-2344 (1990); Guoet al. Virology 174: 217-224 (1990); McCune et al. Cell 53:55-67 (1988);Liao et al. J Virol. April; 87(8):4185-201 (2013).

Likewise, the design ofgp140 envelope forms is also well known in theart, along with the various specific changes which give rise to thegp140C (uncleaved envelope), gp140CF and gp140CFI forms. Envelope gp140forms are designed by introducing a stop codon within the gp41 sequence.See Chakrabarti et al. at FIG. 1 .

Envelope gp140C refers to a gp140 HIV-1 envelope design with afunctional deletion of the cleavage (C) site, so that the gp140 envelopeis not cleaved at the furin cleavage site. The specification describescleaved and uncleaved forms, and various furin cleavage sitemodifications that prevent envelope cleavage are known in the art. Insome embodiments of the gp140C form, two of the R residues in and nearthe furin cleavage site are changed to E, e.g., RRVVEREKR (SEQ ID NO: 2)is changed to ERVVEREKE (SEQ ID NO: 3), and is one example of anuncleaved gp140 form. Another example is the gp140C form which has theREKR site (SEQ ID NO: 1) changed to SEKS (SEQ ID NO: 4). See supra forreferences.

Envelope gp140CF refers to a gp140 HIV-1 envelope design with a deletionof the cleavage (C) site and fusion (F) region. Envelope gp140CFI refersto a gp140 HIV-1 envelope design with a deletion of the cleavage (C)site, fusion (F) and immunodominant (I) region in gp41. See Chakrabartiet al. Journal of Virology vol. 76, pp. 5357-5368 (2002) see for exampleFIG. 1 , and Second paragraph in the Introduction on p. 5357; Binley etal. Journal of Virology vol. 76, pp. 2606-2616 (2002) for example atAbstract; Gao et al. Journal of Virology vol. 79, pp. 1154-1163 (2005);Liao et al. Virology vol. 353(2): 268-282 (2006).

In certain embodiments, the envelope design in accordance with thepresent invention involves deletion of residues (e.g., 5-11, 5, 6, 7, 8,9, 10, or 11 amino acids) at the N-terminus. For delta N-terminaldesign, amino acid residues ranging from 4 residues or even fewer to 14residues or even more are deleted. These residues are between thematuration (signal peptide, usually ending with CX, X can be any aminoacid) and “VPVXXXX . . . ”. In case of CH505 T/F Env as an example, 8amino acids (italicized and underlined in the below sequence) weredeleted: MRVMGIQRNYPQWWIWSMLGFWMLMICNGMWVTVYYGVPVWKEAKTTLFCASDAKAYEKEVHNVWATHACVPTDPNPQE . . . (rest of envelope sequence is indicatedas “ . . . ”) (SEQ ID NO: 5). In other embodiments, the delta N-designdescribed for CH505 T/F envelope can be used to make delta N-designs ofother CH505 envelopes. In certain embodiments, the invention relatesgenerally to an immunogen, gp160, gp120 or gp140, without an N-terminalHerpes Simplex gD tag substituted for amino acids of the N-terminus ofgp120, with an HIV leader sequence (or other leader sequence), andwithout the original about 4 to about 25, for example 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 aminoacids of the N-terminus of the envelope (e.g. gp120). See WO2013/006688,e.g. at pages 10-12, the contents of which publication is herebyincorporated by reference in its entirety.

The general strategy of deletion of N-terminal amino acids of envelopesresults in proteins, for example gp120s, expressed in mammalian cellsthat are primarily monomeric, as opposed to dimeric, and, therefore,solves the production and scalability problem of commercial gp120 Envvaccine production. In other embodiments, the amino acid deletions atthe N-terminus result in increased immunogenicity of the envelopes.

In certain embodiments, the invention provides envelope sequences, aminoacid sequences and the corresponding nucleic acids, and in which the V3loop is substituted with the following V3 loop sequenceTRPNNNTRKSIRIGPGQTFY ATGDIIGNIRQAH (SEQ ID NO: 6). This substitution ofthe V3 loop reduced product cleavage and improves protein yield duringrecombinant protein production in CHO cells.

In certain embodiments, the CH505 envelopes will have added certainamino acids to enhance binding of various broad neutralizing antibodies.Such modifications could include but not limited to, mutations at W680Gor modification of glycan sites for enhanced neutralization.

In certain aspects, the invention provides composition and methods whichuse a selection of sequential CH505 Envs, as gp120s, gp 140s cleaved anduncleaved, gp145s, gp150s and gp160s, stabilized and/or multimerizedtrimers, as proteins, DNAs, RNAs, or any combination thereof,administered as primes and boosts to elicit immune response. SequentialCH505 Envs as proteins would be co-administered with nucleic acidvectors containing Envs to amplify antibody induction. In certainembodiments, the compositions and methods include any immunogenic HIV-1sequences to give the best coverage for T cell help and cytotoxic T cellinduction. In certain embodiments, the compositions and methods includemosaic and/or consensus HIV-1 genes to give the best coverage for T cellhelp and cytotoxic T cell induction. In certain embodiments, thecompositions and methods include mosaic group M and/or consensus genesto give the best coverage for T cell help and cytotoxic T cellinduction. In some embodiments, the mosaic genes are any suitable genefrom the HIV-1 genome. In some embodiments, the mosaic genes are Envgenes, Gag genes, Pol genes, Nef genes, or any combination thereof. Seee.g. U.S. Pat. No. 7,951,377. In some embodiments the mosaic genes arebivalent mosaics. In some embodiments the mosaic genes are trivalent. Insome embodiments, the mosaic genes are administered in a suitable vectorwith each immunization with Env gene inserts in a suitable vector and/oras a protein. In some embodiments, the mosaic genes, for example asbivalent mosaic Gag group M consensus genes, are administered in asuitable vector, for example but not limited to HSV2, would beadministered with each immunization with Env gene inserts in a suitablevector, for example but not limited to HSV-2.

In certain aspects the invention provides compositions and methods ofEnv genetic immunization either alone or with Env proteins to recreatethe swarms of evolved viruses that have led to bnAb induction.Nucleotide-based vaccines offer a flexible vector format to immunizeagainst virtually any protein antigen. Currently, two types of geneticvaccination are available for testing—DNAs and mRNAs.

In certain aspects the invention contemplates using immunogeniccompositions wherein immunogens are delivered as DNA. See Graham B S,Enama M E, Nason M C, Gordon I J, Peel S A, et al. (2013) DNA VaccineDelivered by a Needle-Free Injection Device Improves Potency of Primingfor Antibody and CD8+ T-Cell Responses after rAd5 Boost in a RandomizedClinical Trial. PLoS ONE 8(4): e59340, page 9. Various technologies fordelivery of nucleic acids, as DNA and/or RNA, so as to elicit immuneresponse, both T-cell and humoral responses, are known in the art andare under developments. In certain embodiments, DNA can be delivered asnaked DNA. In certain embodiments, DNA is formulated for delivery by agene gun. In certain embodiments, DNA is administered byelectroporation, or by a needle-free injection technologies, for examplebut not limited to Biojector® device. In certain embodiments, the DNA isinserted in vectors. The DNA is delivered using a suitable vector forexpression in mammalian cells. In certain embodiments the nucleic acidsencoding the envelopes are optimized for expression. In certainembodiments DNA is optimized, e.g. codon optimized, for expression. Incertain embodiments the nucleic acids are optimized for expression invectors and/or in mammalian cells. In non-limiting embodiments these arebacterially derived vectors, adenovirus based vectors, rAdenovirus (e.g.Barouch D H, et al. Nature Med. 16: 319-23, 2010), recombinantmycobacteria (e.g. rBCG or M smegmatis) (Yu, J S et al. Clinical VaccineImmunol. 14: 886-093, 2007; ibid 13: 1204-11, 2006), and recombinantvaccinia type of vectors (Santra S. Nature Med. 16: 324-8, 2010), forexample but not limited to ALVAC, replicating (Kibler K V et al., PLoSOne 6: e25674, 2011 Nov. 9.) and non-replicating (Perreau M et al. J.virology 85: 9854-62, 2011) NYVAC, modified vaccinia Ankara (MVA)),adeno-associated virus, Venezuelan equine encephalitis (VEE) replicons,Herpes Simplex Virus vectors, and other suitable vectors.

In certain aspects the invention contemplates using immunogeniccompositions wherein immunogens are delivered as DNA or RNA in suitableformulations. Various technologies which contemplate using DNA or RNA,or may use complexes of nucleic acid molecules and other entities to beused in immunization. In certain embodiments, DNA or RNA is administeredas nanoparticles consisting of low dose antigen-encoding DNA formulatedwith a block copolymer (amphiphilic block copolymer 704). See Cany etal., Journal of Hepatology 2011 vol. 54 j 115-121; Arnaoty et al.,Chapter 17 in Yves Bigot (ed.), Mobile Genetic Elements: Protocols andGenomic Applications, Methods in Molecular Biology, vol. 859, pp 293-305(2012); Arnaoty et al. (2013) Mol Genet Genomics. 2013 August;288(7-8):347-63. Nanocarrier technologies called Nanotaxi® forimmunogenic macromolecules (DNA, RNA, Protein) delivery are underdevelopment. See for example technologies developed by incellart.

In certain aspects the invention contemplates using immunogeniccompositions wherein immunogens are delivered as recombinant proteins.Various methods for production and purification of recombinant proteins,including trimers such as but not limited to SOSIP based trimers,suitable for use in immunization are known in the art. In certainembodiments recombinant proteins are produced in CHO cells.

The immunogenic envelopes can also be administered as a protein boost incombination with a variety of nucleic acid envelope primes (e.g., HIV-1Envs delivered as DNA expressed in viral or bacterial vectors).

Dosing of proteins and nucleic acids can be readily determined by askilled artisan. A single dose of nucleic acid can range from a fewnanograms (ng) to a few micrograms (μg) or milligram of a singleimmunogenic nucleic acid. Recombinant protein dose can range from a fewμg micrograms to a few hundred micrograms, or milligrams of a singleimmunogenic polypeptide.

Administration: The compositions can be formulated with appropriatecarriers using known techniques to yield compositions suitable forvarious routes of administration. In certain embodiments thecompositions are delivered via intramascular (IM), via subcutaneous, viaintravenous, via nasal, via mucosal routes, or any other suitable routeof immunization.

The compositions can be formulated with appropriate carriers andadjuvants using techniques to yield compositions suitable forimmunization. The compositions can include an adjuvant, such as, forexample but not limited to, alum, poly IC, MF-59 or other squalene-basedadjuvant, ASOIB, or other liposomal based adjuvant suitable for proteinor nucleic acid immunization. In certain embodiments, the adjuvant isGSK AS01E adjuvant containing MPL and QS21. This adjuvant has been shownby GSK to be as potent as the similar adjuvant AS01B but to be lessreactogenic using HBsAg as vaccine antigen [Leroux-Roels et al., IABSConference, April 2013]. In certain embodiments, TLR agonists are usedas adjuvants. In other embodiment, adjuvants which break immunetolerance are included in the immunogenic compositions.

In certain embodiments, the compositions and methods comprise anysuitable agent or immune modulation which could modulate mechanisms ofhost immune tolerance and release of the induced antibodies. Innon-limiting embodiments modulation includes PD-1 blockade; T regulatorycell depletion; CD40L hyperstimulation; soluble antigen administration,wherein the soluble antigen is designed such that the soluble agenteliminates B cells targeting dominant epitopes, or a combinationthereof. In certain embodiments, an immunomodulatory agent isadministered in at time and in an amount sufficient for transientmodulation of the subject's immune response so as to induce an immuneresponse which comprises broad neutralizing antibodies against HIV-1envelope. Non-limiting examples of such agents is any one of the agentsdescribed herein: e.g. chloroquine (CQ), PTP1B Inhibitor—CAS765317-72-4—Calbiochem or MSI 1436 clodronate or any otherbisphosphonate; a Foxo1 inhibitor, e.g. 344355|Foxo1 Inhibitor,AS1842856—Calbiochem; Gleevac, anti-CD25 antibody, anti-CCR4 Ab, anagent which binds to a B cell receptor for a dominant HIV-1 envelopeepitope, or any combination thereof. In non-limiting embodiments, themodulation includes administering an anti-CTLA4 antibody. Non-limitingexamples are ipilimumab and tremelimumab. In certain embodiments, themethods comprise administering a second immunomodulatory agent, whereinthe second and first immunomodulatory agents are different.

There are various host mechanisms that control bNAbs. For example highlysomatically mutated antibodies become autoreactive and/or less fit(Immunity 8: 751, 1998; PloS Comp. Biol. 6 e1000800, 2010; J. Thoret.Biol. 164:37, 1993); Polyreactive/autoreactive naïve B cell receptors(unmutated common ancestors of clonal lineages) can lead to deletion ofAb precursors (Nature 373: 252, 1995; PNAS 107: 181, 2010; J. Immunol.187: 3785, 2011); Abs with long HCDR3 can be limited by tolerancedeletion (JI 162: 6060, 1999; JCI 108: 879, 2001). BnAb knock-in mousemodels are providing insights into the various mechanisms of tolerancecontrol of MPER BnAb induction (deletion, anergy, receptor editing).Other variations of tolerance control likely will be operative inlimiting BnAbs with long HCDR3s, high levels of somatic hypermutations.

Various antibodies names are used throughout the application. Below islisting of antibodies names correlation: CH490=CH235.6; CH491=CH235.7;CH492=CH235.8; CH493=CH235.9; CH555=CH235.10; CH556=CH235.11;CH557=CH235.12. CH and DH prefixes are used interchangeably, e.g. CH235and DH235.

TABLE 1A Summary of CH505 proteins and sequences. (1) See WO2014042669(e.g. at FIG. 17). All of the listed envelopes are designed to includeG458Mut, have glycosylation profile similar to the glycosylation profileof envelopes grown in GnTI^(−/−) cells, or have both modifications. Forspecific non-limiting embodiments of G458Y envelope designs see interalia Example 10, FIGS. 59, 80-82. chim.6R. chim.6R.DS. CHIM.6R. gp120SOSIP.664 SOSIP.664 SOSIP.664V4.1 CHIM.6R. gp160 delta8 gp145 (SOSIP.I)(SIOSIP.II) (SOSIP.III) SOSIP.664V4.2 CH505 TF aa (1) (1) FIG. 23A FIG.23A FIG. 23A FIG. 23A One (1) (1) embodiment of a nucleic acid W53.16(1) (1) FIG. 23A FIG. 23A FIG. 23A FIG. 23A One (1) (1) embodiment of anucleic acid W78.33 (1) (1) FIG. 23A FIG. 23A One (1) (1) embodiment ofa nucleic acid W100.B6 (1) (1) FIG. 23A FIG. 23A One (1) (1) embodimentof a nucleic acid M5 aa FIG. 17B FIG. 17A FIG. 19A FIG. 23A FIG. 23A Oneembodiment of a nucleic acid M11 aa FIG. 17B FIG. 19A FIG. 19A FIG. 23AFIG. 23A One embodiment of a nucleic acid W20.14 aa FIG. 17B FIG. 19AFIG. 19A FIG. 23A FIG. 23A One embodiment of a nucleic acid W30.20 aaFIG. 17B FIG. 19A FIG. 19A FIG. 23A FIG. 23A One embodiment of a nucleicacid W30.12 aa FIG. 17B FIG. 19A FIG. 19A FIG. 23A FIG. 23A Oneembodiment of a nucleic acid W136.B18 aa FIG. 19B FIG. 19A FIG. 19A FIG.23A FIG. 23A One embodiment of a nucleic acid W30.25 aa FIG. 17A FIG.23A FIG. 23A One FIG. 17B embodiment of a nucleic acid W053.25 aa FIG.17A FIG. 23A FIG. 23A One FIG. 17B embodiment of a nucleic acid W053.29aa FIG. 17A FIG. 23A FIG. 23A One FIG. 17B embodiment of a nucleic acid

TABLE 1B Summary of various trimer designs for CH505 M5 G458Y envelopesOne embodiment of One Amino acid sequence embodiment CH505 M5 of nucleicDesign CH505 M5 G458Y acid sequence gp160 FIG. 17B FIG. 81 FIG. 82gp120; FIG. 81 gp120delta8 FIG. 17A FIG. 59C #3 FIG. 59D gp140 FIG. 81gp145 FIG. 19A chim.6R.SOSIP.664 FIG. 81; (SOSIP.I) chim.6R.DS.SOSIP.664FIG. 59C #5 FIG. 59D (SIOSIP.II) CHIM.6R.SOSIP.664V4.1 FIG. 23A FIG. 81;FIG. 59D (SOSIP.III) 59C #1 CHIM.6R.SOSIP.664V4.2 FIG. 23ACHIM.6R.SOSIP.664V4.1.1 FIG. 59C #8 FIG. 59D (aka A73C A561C to formanother S—S bond) chim.6R.SOSIP.664v5.2.8 FIG. 59C #6 FIG. 59Dchim.6R.SOSIP.664v4.1 FIG. 59C #4 FIG. 59D ferritinchim.6R.SOSIP.664v4.1avi FIG. 59C #7 FIG. 59D #CHIM.6R.SOSIP.664V4.2design includes a mutation of the amino acid sequence corresponding toposition 66 of HXB2 sequence. A skilled artisan can readily incorporatethis mutation in any other envelope design, including but not limited toCH505 M5 G458.

It is readily understood that the envelope glycoproteins referenced invarious examples and figures comprise a signal/leader sequence. It iswell known in the art that HIV-1 envelope glycoprotein is a secretoryprotein with a signal or leader peptide sequence that is removed duringprocessing and recombinant expression (without removal of the signalpeptide, the protein is not secreted). See for example Li et al. Controlof expression, glycosylation, and secretion of HIV-1 gp120 by homologousand heterologous signal sequences. Virology 204(1):266-78 (1994) (“Li etal. 1994”), at first paragraph, and Li et al. Effects of inefficientcleavage of the signal sequence of HIV-1 gp120 on its association withcalnexin, folding, and intracellular transport. PNAS 93:9606-9611 (1996)(“Li et al. 1996”), at 9609. Any suitable signal sequence could be used.In some embodiments the leader sequence is the endogenous leadersequence. Most of the gp120 and gp160 amino acid sequences include theendogenous leader sequence. In other non-limiting examples the leaderssequence is human Tissue Plasminogen Activator (TPA) sequence, human CD5leader sequence (e.g. MPMGSLQPLATLYLLGMLVASVLA (SEQ ID NO: 7)). Most ofthe chimeric designs include CD5 leader sequence. A skilled artisanappreciates that when used as immunogens, and for example whenrecombinantly produced, the amino acid sequences of these proteins donot comprise the leader peptide sequences.

Nomenclature for trimers: chim.6R.DS.SOSIP.664 is SOSIP.ICHIM.6R.SOSIP.664 is SOSIP.II; CHIM.6R.SOSIP.664V4.1 is SOSIP.III.Additional trimer designs are listed inter alia in Tables 1A-B, FIGS.23, 59 and 81-82 .

The specific mutations in any one of the designs could be incorporatedin any suitable envelope. For example, using as a guide the CH505 T/Fdesigns in Tables 1A-B, CH505 M envelope can be designed as any trimer.

The invention provides various envelopes and selection of envelopes foruse as immunoges, wherein the various envelope sequences and designfurther comprise change of amino acid position 458 form a Gly (G) to alarge amino acid, e.g. but not limited to G458Y, and wherein in someembodiments the envelope has a glycosylation profile similar to theglycosylation profile of an envelope grown in GnTI^(−/−) cells. Aminoacid position G458 is with reference to the CH505 T/F envelope and askilled artisan can readily determine the corresponding position andamino acid in other envelopes. Any one of the envelopes of the inventioncould be designed and expressed as described in the specification.

The invention is described in the following non-limiting examples.

EXAMPLES Example 1

HIV-1 sequences, including envelopes, and antibodies from HIV-1 infectedindividual CH505 were isolated as described in Liao et al. (2013) Nature496, 469-476 including supplementary materials; See also Gao et al.(2014) Cell 158:481-491.

Recombinant HIV-1 Proteins

HIV-1 Env genes for subtype B, 63521, subtype C, 1086, and subtypeCRF_01, 427299, as well as subtype C, CH505 autologoustransmitted/founder Env were obtained from acutely infected HIV-1subjects by single genome amplification, codon-optimized by using thecodon usage of highly expressed human housekeeping genes, de novosynthesized (GeneScript) as gp140 or gp120 (AE.427299) and cloned into amammalian expression plasmid pcDNA3.1/hygromycin (Invitrogen).Recombinant Env glycoproteins were produced in 293F cells cultured inserum-free medium and transfected with the HIV-1 gp140- orgp120-expressing pcDNA3.1 plasmids, purified from the supernatants oftransfected 293F cells by using Galanthus nivalis lectin-agarose (VectorLabs) column chromatography, and stored at −80° C. Select Env proteinsmade as CH505 transmitted/founder Env were further purified by superose6 column chromatography to trimeric forms, and used in binding assaysthat showed similar results as with the lectin-purified oligomers.

ELISA

Binding of patient plasma antibodies and CH103, and DH235(CH235), SeeGao et al. (2014) Cell 158:481-491, clonal lineage antibodies toautologous and heterologous HIV-1 Env proteins was measured by ELISA asdescribed previously. Plasma samples in serial threefold dilutionsstarting at 1:30 to 1:521,4470 or purified monoclonal antibodies inserial threefold dilutions starting at 100 μg ml-1 to 0.000 μg ml-1diluted in PBS were assayed for binding to autologous and heterologousHIV-1 Env proteins. Binding of biotin-labelled CH103 at thesubsaturating concentration was assayed for cross-competition byunlabeled HIV-1 antibodies and soluble CD4-Ig in serial fourfolddilutions starting at 10 μg ml-1. The half-maximal effectiveconcentration (EC50) of plasma samples and monoclonal antibodies toHIV-1 Env proteins were determined and expressed as either thereciprocal dilution of the plasma samples or concentration of monoclonalantibodies.

Surface Plasmon Resonance Affinity and Kinetics Measurements

Binding Kd and rate constant (association rate (Ka)) measurements ofmonoclonal antibodies and all candidate UCAs to the autologous Env C.CH05 gp140 and/or the heterologous Env B.63521 gp120 are carried out onBlAcore 3000 instruments as described previously. Anti-human IgG Fcantibody (Sigma Chemicals) is immobilized on a CMS sensor chip to about15,000 response units and each antibody is captured to about 50-200response units on three individual flow cells for replicate analysis, inaddition to having one flow cell captured with the control Synagis(anti-RSV) monoclonal antibody on the same sensor chip. Doublereferencing for each monoclonal antibody—HIV-1 Env binding interactionsis used to subtract nonspecific binding and signal drift of the Envproteins to the control surface and blank buffer flow, respectively.Antibody capture level on the sensor surface is optimized for eachmonoclonal antibody to minimize rebinding and any associated avidityeffects. C.CH505 Env gp140 protein is injected at concentrations rangingfrom 2 to 25 μg ml-1, and B.63521 gp120 was injected at 50-400 μg ml-1for UCAs and early intermediates IA8 and IA4, 10-100 μg ml-1 forintermediate IA3, and 1-25 μg ml-1 for the distal and mature monoclonalantibodies. All curve-fitting analyses are performed using global fit ofto the 1:1 Langmuir model and are representative of at least threemeasurements. All data analysis was performed using the BlAevaluation4.1 analysis software (GE Healthcare).

Neutralization Assays

Neutralizing antibody assays in TZM-bl cells are performed as describedpreviously. Neutralizing activity of plasma samples in eight serialthreefold dilutions starting at 1:20 dilution and for recombinantmonoclonal antibodies in eight serial threefold dilutions starting at 50μg ml-1 are tested against autologous and herologous HIV-1Env-pseudotyped viruses in TZM-bl-based neutralization assays using themethods known in the art. Neutralization breadth of CH103 is determinedusing a panel of 196 of geographically and genetically diverseEnv-pseudoviruses representing the major circulated genetic subtypes andcirculating recombinant forms. HIV-1 subtype robustness is derived fromthe analysis of HIV-1 clades over time. The data are calculated as areduction in luminescence units compared with control wells, andreported as IC50 in either reciprocal dilution for plasma samples or inmicrograms per microlitre for monoclonal antibodies.

The GenBank accession numbers for 292 CH505 Env proteins areKC247375-KC247667, and accessions for 459 V_(H)DJ_(H) and 174 V_(L)J_(L)sequences of antibody members in the CH103 clonal lineage areKC575845-KC576303 and KC576304-KC576477, respectively.

Example 2

Binding of Sequential Envelopes to CH103 and CH235 CD4 Binding Site bnAbLineages Members.

The binding assay was an ELISA with the envelope protein bound to thewell surface of a 96 well plate, and the antibody in questions incubatedwith the envelope bound to the plate. After washing, an enzyme-labeledanti-human IgG antibody was added and after incubation, washed away. Theintensity of binding was determined by the intensity of enzyme-activatedcolor in the well.

TABLE 2 ELISA binding, log-transformed area under the curve (AUC) valuesfor a realization with four Env-derived gp120 antigens, assayed againstmembers of the CH103 bnAb lineage from universal ancestor (UCA), throughintermediate ancestors (IA8-IA1) to the mature bnAb. Values of 0indicate no binding. The transmitted-founder (TF) antigen was derivedfrom Env w004.3. Antigen UCA IA8 IA7 IA6 IA4 IA3 CH105 IA2 CH104 IA1CH106 CH103 TF 3.5 5.5 9.2 9.1 10.1 11 11.2 10.8 10.4 10.4 11.3 12.6w053.16 0 0 0 0 0.2 1.1 9 9.3 9.9 8.8 9.8 11.6 w078.33 0 0 0 0 0 0 8.9 99 8.2 9.5 11.1 w100.B6 0 0 0 0 0 0 11 12.1 11 12.2 11.8 7.1

TABLE 3 ELISA binding, log-transformed area under the curve (AUC) valuesfor a realization with five Env-derived gp120 antigens, assayed againstmembers of the CH103 bnAb lineage from universal ancestor (UCA), throughintermediate ancestors (IA8-IA1) to the mature bnAb. Values of 0indicate no binding. Antigen names beginning with M were synthesized bysite-directed mutagenesis. Antigen UCA IA8 IA7 IA6 IA4 IA3 CH105 IA2CH104 IA1 CH106 CH103 M11 2.6 6.2 10.1 10 10.5 11.8 11.7 12.7 12 12.212.8 13.4 M5 0 0.6 2.3 3.3 3.8 6.8 8.6 7.8 9 7 8.4 9.8 w020.14 0.3 3.47.2 7.9 8.6 9.5 10.4 11 10.4 10.3 11.2 12.6 w030.28 0 1.6 3.5 6.3 6.57.7 9.1 11.1 10.7 10.1 11.7 12.8 w078.15 0 0 0.7 1 1.3 3 10.1 11.5 10.810.9 11 10.7 w053.31 0 0 0 0 0 0 13.5 13.3 13.7 13.4 13.4 13.6

TABLE 4 ELISA binding, log-transformed area under the curve (AUC) valuesfor a realization with five Env-derived gp120 antigens, assayed againstmembers of the DH235 (CH235) bnAb helper lineage from universal ancestor(UCA), through intermediate ancestors (I4-I1) to mature bnAbs. Values of0 indicate no binding. Antigen names beginning with M were synthesizedby site-directed mutagenesis. Antigen UCA I4 I3 I2 I1 DH235 CH236 CH239CH240 CH241 M11 0 0 0 0 2.8 7.6 1.4 1.4 0.5 9.7 M5 0.2 1.4 7 6.9 9.211.4 7.3 12.9 7.4 14.5 w020.14 0 0 2.7 1.2 6.5 9.9 6.7 9 3.8 13.1w030.28 0 0 0 0 2.4 6.7 1.5 3.6 0.3 9.6 w078.15 0 0 0 0 0 0 0 0 0 0w053.31 0 0 0 0 0 1.1 0 0 0 1.4

TABLE 5 ELISA binding, log-transformed area under the curve (AUC) valuesfor a realization that embodies ten Env-derived gp120 antigens, assayedagainst members of the CH103 bnAb lineage from universal ancestor (UCA),through intermediate ancestors (IA8-IA1) to the mature bnAb. Values of 0indicate no binding. Antigen names beginning with M were synthesized bysite-directed mutagenesis. Antigen UCA IA8 IA7 IA6 IA4 IA3 CH105 IA2CH104 IA1 CH106 CH103 M11 2.6 6.2 10.1 10 10.5 11.8 11.7 12.7 12 12.212.8 13.4 M5 0 0.6 2.3 3.3 3.8 6.8 8.6 7.8 9 7 8.4 9.8 w020.14 0.3 3.47.2 7.9 8.6 9.5 10.4 11 10.4 10.3 11.2 12.6 w030.28 0 1.6 3.5 6.3 6.57.7 9.1 11.1 10.7 10.1 11.7 12.8 w078.15 0 0 0.7 1 1.3 3 10.1 11.5 10.810.9 11 10.7 w053.16 0 0 0 0 0.2 1.1 9 9.3 9.9 8.8 9.8 11.6 w030.21 0 00 0 0 0 10.6 11.5 11.3 11.8 10.9 12.2 w078.33 0 0 0 0 0 0 8.9 9 9 8.29.5 11.1 w100.B6 0 0 0 0 0 0 11 12.1 11 12.2 11.8 7.1 w053.31 0 0 0 0 00 13.5 13.3 13.7 13.4 13.4 13.6

Example 3

Combinations of Antigens Derived from CH505 Envelope Sequences for SwarmImmunizations

Provided herein are non-limiting examples of combinations of antigensderived from CH505 envelope sequences for a swarm immunization. Withoutlimitations, these selected combinations comprise envelopes whichprovide representation of the sequence and antigenic diversity of theHIV-1 envelope variants which lead to the induction and maturation ofthe CH103 and CH235 antibody lineages. The identification of bnAblineage (CH103) and envelopes which bind preferentially to variousmembers of this lineage provides a direct strategy for the selection ofEnvs (out of millions possible envelopes naturally occurring in an HIV-1infected individual) that might have engaged UCA and participated inbnAb development, and thus could serve as immunogens in a vaccineformulation. The identification of helper lineage (CH235) and envelopeswhich bind preferentially to various members this lineage provides adirect strategy for the selection of Envs (out of millions possibleenvelopes naturally occurring in an HIV-1 infected individual) thatmight have engaged UCA and participated in bnAb development, and thuscould serve as immunogens in a vaccine formulation.

The selection includes priming with a virus which binds to the UCA, forexample a T/F virus or another early (e.g. but not limited to week004.3, or 004.26) virus envelope. In certain embodiments the prime couldinclude D-loop variants. In certain embodiments the boost could includeD-loop variants. In certain embodiments, these D-loop variants areenvelope escape mutants not recognized by the UCA. Non-limiting examplesof such D-loop variants are envelopes designated as M10, M11, M19, M20,M21, M5, M6, M7, M8, M9, M14 (TF_M14), M24 (TF_24), M15, M16, M17, M18,M22, M23, M24, M25, M26. See Gao et al. (2014) Cell 158:481-491.

Non-limiting embodiments of envelopes selected for swarm vaccination areshown as the selections described below. A skilled artisan wouldappreciate that a vaccination protocol can include a sequentialimmunization starting with the “prime” envelope(s) and followed bysequential boosts, which include individual envelopes or combination ofenvelopes. In another vaccination protocol, the sequential immunizationstarts with the “prime” envelope(s) and is followed with boosts ofcumulative prime and/or boost envelopes. In certain embodiments, thesequential immunization starts with the “prime” envelope(s) and isfollowed by boost(s) with all or various combinations of the envelopesin the selection. In certain embodiments, the prime does not include T/Fsequence (W000.TF). In certain embodiments, the prime includes w004.03envelope. In certain embodiments, the prime includes w004.26 envelope.In certain embodiment the prime includes M11. In certain embodiments theprime includes M5. In certain embodiments, the immunization methods donot include immunization with HIV-1 envelope T/F. In certainembodiments, the immunization methods do not include a schedule of fourvalent immunization with HIV-1 envelopes T/F, w053.16, w078.33, andw100.B6.

In certain embodiments, there is some variance in the immunizationregimen; in some embodiments, the selection of HIV-1 envelopes may begrouped in various combinations of primes and boosts, either as nucleicacids, proteins, or combinations thereof.

In certain embodiments the immunization includes a prime administered asDNA, and MVA boosts. See Goepfert, et al. 2014; “Specificity and 6-MonthDurability of Immune Responses Induced by DNA and Recombinant ModifiedVaccinia Ankara Vaccines Expressing HIV-1 Virus-Like Particles” J InfectDis. 2014 Feb. 9. [Epub ahead of print].

HIV-1 Envelope selection A (five envelopes): M11; w020.14; w030.28;w078.15; w053.31

HIV-1 Envelope selection B (six envelopes): M11; M5; w020.14; w030.28;w078.15; w053.31

HIV-1 Envelope selection C (ten envelopes): M11; M5; w020.14; w030.28;w078.15; w053.16; w030.21; w078.33; w100.B6; w053.31.

HIV-1 Envelopes selection D (six envelopes): M5, M11, 20.14, 30.28,30.23, 136.B18.

HIV-1 Envelopes selection E (six envelopes): M5, M11, 20.14, 30.20,30.23, 136.B18.

HIV-1 Envelopes selection F (six envelopes—P186 study): M5, M11, 20.14,30.20, 30.12, 136.B18.

HIV-1 envelope selection G (EnvSeq-2): M5, 30.25; 53.25; 53.29.

HIV-1 envelope selection H (EnvSeq-3): M5, 30.20; 20.14, 30.12.

HIV-1 envelope selection I: T/F, 53.16, optionally 78.33, 100.B6, or anyother suitable envelope, wherein each envelope comprises G458mutation,e.g. G458Y.

Selections using M5 as a prime, e.g. but not limited to D, E, F, G or Hare expected to engage receptors and drive progression of CH235 lineageof antibodies.

The selections of CH505-Envs were down-selected from a series of 400CH505 Envs isolated by single-genome amplification followed for 3 yearsafter acute infection, based on experimental data. The enhancedneutralization breadth that developed in the CD4-binding site (bs) CH103antibody lineage that arose in subject CH505 developed in conjunctionwith epitope diversification in the CH505's viral quasispecies. It wasobserved that at 6 months post-infection there was more diversificationin the CD4bs epitope region in this donor than sixteen other acutelyinfected donors. Population breadth did not arise in the CH103 antibodylineage until the epitope began to diversify. A hypothesis is that theCH103 linage drove viral escape, but then the antibody adapted to therelatively resistant viral variants. As this series of events wasrepeated, the emerging antibodies evolved to tolerate greater levels ofdiversity in relevant sites, and began to be able to recognize andneutralize diverse heterologous forms for the virus and manifestpopulation breadth. In certain embodiments, six envs are selected fromCH505 sequences to reflect diverse variants for making Envpseudoviruses, with the goal of recapitulating CH505 HIV-1 antigenicdiversity over time, making sure selected site (i.e. those sitesreflecting major antigenic shifts) diversity was represented.

Specifically, for CH505 the virus and envelope evolution were mapped,and the CH103 CD4 binding-site bnAb evolution. In addition, 135 CH505varied envelope pseudotyped viruses were made and tested them forneutralization sensitivity by members of the CH103 bnAb lineage (e.g,FIG. 3 ). From this large dataset, in one embodiment, six Env variantswere chosen for immunization based on sequence diversity, and antigenicdiversity, for example binding to antibodies in the CH103 and/or CH235lineage (Tables 3-5).

In certain embodiments, the envelopes are selected based on Env mutantswith sites under diversifying selection, in which thetransmitted/founder (T/F) Env form vanished below 20% in any sample,i.e. escape variants; signature sites based on autologous neutralizationdata, i.e. Envs with statistically supported signatures for escape frommembers of the CH103 bnAb lineage; and sites with mutations at thecontact sites of the CH103 antibody and HIV Env. In this manner, asequential swarm of Envs was selected for immunization to represent theprogression of virus escape mutants that evolved during bnAb inductionand increasing neutralization breadth in the CH505 donor.

In certain embodiments, additional sequences are selected to containfive additional specific amino acid signatures of resistance that wereidentified at the global population level. These sequences containstatistically defined resistance signatures, which are common at thepopulation level and enriched among heterologous viruses that CH103fails to neutralize. When they were introduced into the TF sequence,they were experimentally shown to confer partial resistance toantibodies in the CH103 lineage. Following the reasoning that serialviral escape and antibody adaptation to escape is what ultimate selectsfor neutralizing antibodies that exhibit breadth and potency againstdiverse variants, in certain embodiments, inclusion of these variants ina vaccine may extend the breadth of vaccine-elicited antibodies evenbeyond that of the CH103 lineage. Thus the overarching goal will be totrigger a CH103-like lineage first using the CH505TF modified M11, thatis well recognized by early CH103 ancestral states, then vaccinatingwith antigenic variants, to allow the antibody lineage to adapt throughsomatic mutation to accommodate the natural variants that arose inCH505. In certain embodiments, vaccination regimens include a total offive sequences (Selection A) that capture the antigenic diversity ofCH505. In another embodiment, additional antigenic diversity is added(Selection B and C), to enable the induction of antibodies byvaccination that may have even greater breadth than those antibodiesisolated from CH505.

In some embodiments, the CH505 sequences that represent the accumulationof viral sequence and antigenic diversity in the CD4bs epitope of CH103in subject CH505 are represented by selection A, selection B, orselection C.

M11 is a mutant generated to include two mutations in the loop D(N279D+V281G relative to the TF sequence) that enhanced binding to theCH103 lineage. These were early escape mutations for another CD4bsautologous neutralizing antibody lineage, but might have served topromote early expansion of the CH103 lineage.

In certain embodiments, the two CH103 resistance signature-mutationsequences added to the antigenic swarm are: M14 (TF with S364P), and M24(TF with S375H+T202K+L520F+G459E). They confer partial resistance to theTF with respect to the CH103 lineage. In certain embodiments, theseD-loop mutants are administered in the boost.

Example 4

Immunization Protocols in Subjects with Swarms of HIV-1 Envelopes.

Immunization protocols contemplated by the invention include envelopessequences as described herein including but not limited to nucleic acidsand/or amino acid sequences of gp160s, gp150s, gp145, cleaved anduncleaved gp140s, stabilized trimers, e.g. but not limited to SOSIPtrimers, gp120s, gp41s, N-terminal deletion variants as describedherein, cleavage resistant variants as described herein, or codonoptimized sequences thereof. A skilled artisan can readily modify thegp160 and gp120 sequences described herein to obtain these envelopevariants. The swarm immunization selections can be administered in anysubject, for example monkeys, mice, guinea pigs, or human subjects.

In non-limiting embodiments, the immunization includes a nucleic acidwhich is administered as DNA, for example in a modified vaccinia vector(MVA). In non-limiting embodiments, the nucleic acids encode gp160envelopes. In other embodiments, the nucleic acids encode gp120envelopes. In other embodiments, the boost comprises a recombinant gp120envelope. The vaccination protocols include envelopes formulated in asuitable carrier and/or adjuvant, for example but not limited to alum.In certain embodiments the immunizations include a prime, as a nucleicacid or a recombinant protein, followed by a boost, as a nucleic acid ora recombinant protein. A skilled artisan can readily determine thenumber of boosts and intervals between boosts.

In some embodiments, the immunization methods comprise immunizationprime with a nucleic acid, for example but not limited to priming twotimes with DNA. In some embodiments the nucleic acid prime isadministered one, two, three or four times. In some embodiments the twoDNA prime is administered via electroporation (DNA-EP). In someembodiments, the primer and boost is administered as RNA. The primes arefollowed by boost with sequential envelopes. The boosting envelopescould be in any suitable form, e.g. but not limited to gp140s, assoluble or stabilized SOSIP trimers.

Table 6 shows a non-limiting example of an immunization protocol using aselection of HIV-1 envelopes

Envelope Prime Boost(s) Boost(s) Boost(s) M11 M11 as a nucleic acid e.g.DNA/MVA vector and/ or protein W020.14 W020.14 as a nucleic acid e.g.DNA/MVA and/or protein W030.28 W030.28 as nucleic acid e.g. DNA/MVAand/or protein W078.15 w078.15 as nucleic acid e.g. DNA/MVA and/orprotein W100.B6 W100.B6 as nucleic acid e.g. DNA/MVA and/or protein

Table 7 shows a non-limiting example of an immunization protocol using aselection of HIV-1 envelopes

Envelope Prime Boost(s) Boost(s) Boost(s) M11 M11 as a M11 as a M11 as aM11 as a nucleic acid nucleic acid nucleic acid nucleic acid e.g. e.g.e.g. e.g. DNA/MVA DNA/MVA DNA/MVA DNA/MVA vector and/ vector and/orvector and/or vector and/or or protein protein protein protein W020.14W020.14 W020.14 W020.14 as a nucleic as a nucleic as a nucleic acid e.g.acid e.g. acid e.g. DNA/MVA DNA/MVA DNA/MVA and/or protein and/orprotein and/or protein W030.28 W030.28 W030.28 as nucleic as nucleicacid e.g. acid e.g. DNA/MVA DNA/MVA and/or protein and/or proteinW078.15 w078.15 w078.15 as nucleic as nucleic acid e.g. acid e.g.DNA/MVA DNA/MVA and/or protein and/or protein W100.B6 W100.B6 as nucleicacid e.g. DNA/MVA and/or protein

In certain embodiments, after administering a prime with M11, subsequentimmunizations include all other envelopes as nucleic acids and/orproteins.

Table 8 shows a non-limiting example of an immunization protocol using aswarm of HIV-1 envelopes

Prime/ Envelope Prime Boost Boost(s) Boost(s) Boost(s) M11 M11 as anucleic acid e.g. DNA/ MVA vector and/ or protein M5 M5 as a nucleicacid e.g. DNA/ MVA and/ or protein W020.14 W020.14 as a nucleic acide.g. DNA/ MVA and/ or protein W030.28 W030.28 as nucleic acid e.g. DNA/MVA and/ or protein W078.15 w078.15 as nucleic acid e.g. DNA/ MVA and/or protein W100.B6 W100.B6 as nucleic acid e.g. DNA/ MVA and/ or protein

Table 9 shows a non-limiting example of an immunization protocol using aswarm of HIV-1 envelopes

Prime/ Envelope Prime Boost Boost(s) Boost(s) Boost(s) M11 M11 as a M11as a M11 as a M11 as a M11 as a nucleic nucleic nucleic nucleic nucleicacid e.g. acid e.g. acid e.g. acid e.g. acid e.g. DNA/ DNA/ DNA/ DNA/DNA/ MVA MVA MVA MVA MVA vector vector vector vector vector and/orand/or and/or and/or and/or protein protein protein protein protein M5Optionally M5 as a M5 as a M5 as a M5 as a M5 as a nucleic nucleicnucleic nucleic nucleic acid e.g. acid e.g. acid e.g. acid e.g. acide.g. DNA/ DNA/ DNA/ DNA/ DNA/ MVA MVA MVA MVA MVA and/or and/or and/orand/or and/or protein protein protein protein protein W020.14 W020.14W020.14 W020.14 as a as a as a nucleic nucleic nucleic acid e.g. acide.g. acid e.g. DNA/ DNA/ DNA/ MVA MVA MVA and/or and/or and/or proteinprotein protein W030.28 W030.28 W030.28 as nucleic as nucleic acid e.g.acid e.g. DNA/ DNA/ MVA MVA and/or and/or protein protein W078.15w078.15 w078.15 as nucleic as nucleic acid e.g. acid e.g. DNA/ DNA/ MVAMVA and/or and/or protein protein W100.B6 W100.B6 as nucleic acid e.g.DNA/ MVA and/or protein

In certain embodiments, after administering a prime with M11 andoptionally with M5, subsequent immunizations include all other envelopesas nucleic acids and/or proteins.

Table 10 shows a non-limiting example of immunization protocol using aselection of ten HIV-1 envelopes

Envelope Prime Prime/Boost Boost(s) Boost(s) Boost(s) Boost(s) Boost(s)Boost(s) Boost(s) M11 M11 as a nucleic acid e.g. DNA/ MVA vector and/orprotein M5 M5 as a nucleic acid e.g. DNA/ MVA and/or protein W020.14W020.14 as a nucleic acid e.g. DNA/ MVA and/or protein W030.28 W030.28as nucleic acid e.g. DNA/ MVA and/or protein W078.15 w078.15 as nucleicacid e.g. DNA/ MVA and/or protein W053.16 W053.16 as nucleic acid e.g.DNA/ MVA and/or protein W030.21 W030.21 as nucleic acid e.g. DNA/ MVAand/or protein W078.33 W078.33 as nucleic acid e.g. DNA/ MVA and/orprotein W100.B6 W100.B6 as nucleic acid e.g. DNA/ MVA and/or proteinW053.31 W053.31 as nucleic acid e.g. DNA/ MVA and/or protein

Table 11 shows a non-limiting example of immunization protocol using aselection of six HIV-1 envelopes

Envelope Prime Prime/Boost Boost(s) Boost(s) Boost(s) M11 M11 as a M11as a M11 as a M11 as a M11 as a nucleic nucleic nucleic nucleic nucleicacid e.g. acid e.g. acid e.g. acid e.g. acid e.g. DNA/ DNA/ DNA/ DNA/DNA/ MVA MVA MVA MVA MVA vector vector vector vector vector and/orand/or and/or and/or and/or protein protein protein protein protein M5Optionally M5 as a M5 as a M5 as a M5 as a M5 as a nucleic nucleicnucleic nucleic nucleic acid e.g. acid e.g. acid e.g. acid e.g. acide.g. DNA/ DNA/ DNA/ DNA/ DNA/ MVA MVA MVA MVA MVA and/or and/or and/orand/or and/or protein protein protein protein protein W020.14 W020.14W020.14 W020.14 as a as a as a nucleic nucleic nucleic acid e.g. acide.g. acid e.g. DNA/ DNA/ DNA/ MVA MVA MVA and/or and/or and/or proteinprotein protein W030.20 W030.20 W030.20 as nucleic as nucleic acid e.g.acid e.g. DNA/ DNA/ MVA MVA and/or and/or protein protein W030.12w030.12 w030.12 as nucleic as nucleic acid e.g. acid e.g. DNA/ DNA/ MVAMVA and/or and/or protein protein W136.B18 W136.B18 as nucleic acid e.g.DNA/ MVA and/or protein

Table 12 shows a non-limiting example of immunization protocol using aselection of six HIV-1 envelopes

Envelope Prime Boost(s) Boost(s) Boost(s) Boost(s) M11 M11 as a nucleicacid e.g. DNA/ MVA vector and/or protein M5 Optionally M5 as a nucleicacid e.g. DNA/ MVA and/or protein W020.14 W020.14 as a nucleic acid e.g.DNA/ MVA and/or protein W030.20 W030.20 as nucleic acid e.g. DNA/ MVAand/or protein W030.12 w030.12 as nucleic acid e.g. DNA/ MVA and/orprotein W136.B18 W136.B18 as nucleic acid e.g. DNA/ MVA and/or protein

In certain embodiments, after administering a prime with M11 andoptionally with M5, subsequent immunizations include sequential orcumulative addition of the other envelopes as nucleic acids and/orproteins.

Table 13 shows a non-limiting example of immunization protocol using aselection of four HIV-1 envelopes

Envelope Prime Boost(s) Boost(s) Boost(s) M5 M5as a nucleic acid e.g.DNA/MVA vector and/ or protein W30.25 W30.25 as a nucleic acid e.g.DNA/MVA vector and/ or protein W53.25 W53.25 as a nucleic acid e.g.DNA/MVA vector and/ or protein W53.29 W53.29 as a nucleic acid e.g.DNA/MVA vector and/ or protein

In certain embodiments an immunization protocol could prime with abivalent or trivalent Gag mosaic (Gag 1 and Gag 2, Gag 1, Gag 2 andGag3) in a suitable vector.

Example 5A

Env Mixtures of the CH505 Virus are Expected to Induce the Beginning ofCD4 Binding Site BnAb Lineages CH103 and CH235

The combinations of envelopes described in Examples 2-4 will be testedin any suitable subject. Suitable animal models include withoutlimitation mice, including humanized mice, guinea pigs, or non-humanprimates (NHPs). For example an animal is administered with thefollowing antigens, as DNA and/or proteins, in any suitable for, in thefollowing immunization schedule: loop D mutant M5 and/or M11. That willgive the best CH103 UCA binder (M11) and the best CH235 UCA binder (M5).Immunization 2: week 020.14. Immunization 3: Week 030.28. Immunization4: week 078.15. Immunization 5: week 100.B6. Immunization 5: swarm ofall six envelopes. Adjuvant is a TLR-4 agonist (GLA-syntheticmonophosphoryl lipid A) in stable emulsion from Infectious DiseaseResearch Institute, Seattle Wash.

In another embodiment, the prime is M5 and M11. The boost includes20.14, 30.20, 30.12, and 136.B18, sequentially or additively.

Example 5B

Immunization Elicits Heterologous and Autologous Tier 2 NeutralizingAntibodies.

While improved breadth of vaccine-induced neutralizing antibodyresponses against tier 2 viruses are needed for a protective HIV-1vaccine, elicitation of bnAbs by vaccination has proven challenging.

This example shows elicitation of heterologous and autologous tier 2neutralizing antibodies with sequential Env vaccination in rhesusmacaques. See also FIGS. 25-34 . The method comprised administering T/Fenvelope as gp145 DNA via electroporation, followed by boosting withT/F, w053.16, w078.33 and w100.B6 envelopes administered as gp140Cenvelopes.

Co-evolution studies of the CH103 lineage of antibodies and viruses fromthe same infected person CH505 provides a roadmap for how bnAbs developduring natural infection (Liao et al. Nature 2014; Bonsignori et al.Cell 2016).

This animal study compared the immunogenicity of CH505 gp140C oligomersto CH505-CD40 conjugates. We hypothesize that a roadblock to bnAbinduction by vaccination is the lack of B cell stimulation by antigenpresenting cells (dendritic cells and monocytes), and that bNAbs,similar to those in the CH103 bnAb lineage, can be induced byvaccination with sequential Envs from CH505 (TF, w053.16, w078.33 andw100.B6). In this experiment the T/F envelope was administered as a DNAprime. In some animals the boosting envelopes (TF, w053.16, w078.33 andw100.B6) were administered as gp140C envelopes. In some animals theseenvelopes were targeted to antigen presenting cells by a CD40antibody—human anti-CD40 IgG4 was linked to the CH505 gp140C.

It is possible that the reduced immunogenicity of the anti-CD40IgG4-CH505 Env regimen is due to anti-drug antibodies in rhesusmacaques.

This example shows that: DNA-EP prime and gp140C oligomer boosts inducedautologous tier 2 neutralization in 1 of 4 macaques; heterologous tier 2neutralization of 9/12 tier 2 isolates was also elicited in the samemacaque; and that CD4 binding site directed plasma IgG was present inwildtype Env immunized macaques. RSC3-reactive B cells were sorted frommacaques and the binding and neutralization screening is ongoing.

This example demonstrated that sequential Env immunogens, including thesequential immunogens used in this study could induce heterologous Tier2 neutralizatoin. One alternative to increase the response rate of bnAbinduction is the use of sequential near-native soluble CH505 trimers(e.g. but not limited to SOSIP based trimers as described herein).Immunization with CH505 stabilized trimers while modulating immunetolerance with immune checkpoint inhibitors is also underway.

In some embodiments, the immunization methods could compriseimmunization prime with a nucleic acid, for example but not limited topriming two times with DNA, In some embodiments the nucleic acid primeis administered one, two, three or four times. In some embodiments thetwo DNA prime is administered via electroporation (DNA-EP). In someembodiments the nucleic acid encodes any suitable form of the envelope.In some embodiments, the primer and boost is administered as RNA. Theprimes are followed by boost with sequential envelopes. The boostingenvelopes could be in any suitable form, e.g. but not limited to gp140s,as soluble or stabilized SOSIP trimers, e.g. but not limited toSOSIP.III.

Example 6A

Over the past five years, the HIV vaccine development field has realizedthat immunization with a single HIV envelope protein will not besuccessful at inducing bnAbs^(1,2). Moreover, with evidence for a roleof host immune tolerance control mechanisms in limiting the induction ofbnAbs^(1,3), the biology of bnAbs has begun to be elucidated. The roleof the structure of the Env immunogen is undoubtedly important, as theEnv must contain sufficiently native bnAb epitopes to bind in optimalaffinities to the unmutated common ancestor (UCA, naïve B cellreceptors) of bnAb lineages^(2,4). Thus, the concept of B cell lineageimmunogen design has arisen, whereby lineages of bnAbs are elucidated,and Envs chosen for sequential immunizations based on optimized affinityof Env immunogens for BCRs at sequential steps of the affinitymaturation pathway of bnAb lineages²

While Envs have been designed for reacting with UCAs of heterologousbnAb lineages^(4,5), we have taken the approach of defining, in selectHIV-infected individuals who make bnAbs, the natural sequence of Envsthat induced the bnAb lineages in order to make immunogen down selectionan evidence-based decision. While such immunogens are designed for theUCA and intermediate antibodies of one particular bnAb lineage, theyhold promise for inducing bnAb lineages in multiple individuals becauseof the remarkable conserved usage of VH and VL genes of bnAbs and therestricted nature of antibody motifs for many bnAb types, particularlyfor the gp41 membrane proximal region⁶, the CD4 binding site⁷ and theV1V2-glycan site^(1,8-10).

Two Types of CD4 Binding Site Antibodies

There are several types of CD4 binding site (bs) bnAbs two of which area) heavy chain complementarity determining region 3 (HCDR3) binders andb) CD4 mimicking bnAbs⁷. HCDR3 binding CD4 binding site bnAbs approachthe CD4 binding site with the HCDR3 and other VH and VL loops withmultiple loop-based interactions. Several different VHs and VLs are usedby HCDR3 binding bnAbs with VH3 and VH4 the most common. In contrast,CD4 mimicking bnAbs have restricted VH usage and either use VH1-2*02 orVH1-46. When VH1-2*02 is used, the light chain LCDR3 must be five aminoacids in length. However, when VH1-46 is used, the LCDR3 can be ofnormal (10-13 aa) in length. Both VH1-2*02 and VH1-46 CD4 mimickingantibodies approach the CD4 binding site in a highly homologous mannerto the approach of CD4, and structural analysis of such bnAbsdemonstrates both structural similarity to CD4, as well as nearidentical structures to each of these types of antibodies⁷. Finally,HCDR3 binders are less broad and potent than CD4 mimicking antibodies,with HCDR3 binders neutralizing ˜50% of isolates (e.g., CH103, CH98)while CD4 mimickers neutralizing 90-95% of isolates (e.g., CH235.12,VRC01)⁷. Thus, both types of antibodies are desirable to induce withvaccination as components of a polyclonal bnAb response.

The CH505 African HIV-infected individual that makes both types of CD4bsbnAbs over 6 years (See Liao et al. (2013) Nature 496, 469-476 includingsupplementary materials; See also Gao et al. (2014) Cell 158:481-491;Example 8)

Thus, from African individual CH505, we have isolated both sequentialEnvs and bnAbs over time, and mapped the co-evolution of two bnAblineages, the CH103 CD4 binding site HCDR3 binder bnAb lineage¹¹ and theCH235 CD4 mimicking CD4bs VH1-46 bnAb lineage¹². The CH103 HCDR3 bindertype of CD4 binding site antibody achieved 55% maximum breadth and 4.5mcg inhibitory concentration 50 (IC50) neutralization of cross-cladeHIVs¹¹. In contrast, the CH235 CD4 mimicking CD4 binding site antibodyachieved 90% neutralization and neutralizing IC50 of 0.7 mcg/ml. Here,we will describe the work of development of sequential Env regimens toinduce both of these types of bnAb lineages, and propose here the newsequential Envs to specifically initiate CH235-like CD4 mimicking bnAblineages.

The EnvSeq-1 Sequential Vaccine from CH505 Designed to Induce HCDR3-Typeof CD4 Binding Site bnAbs

We have developed a 4-valent immunogen comprised of CH505 envelopes thathave been designed to trigger the CH103 lineage UCA to clonally expandand start off CH103-like CD4bs HCDR3-binder types of B cell lineages(TF; w053.16; w078.33; w100.B6 the EnvSeq-1 vaccine, see WO2014042669incorporated by reference in its entirety). In SPR assays, thetransmitted/founder (T/F) Env gp120 reacted with the UCA of the CH103lineage with a K_(D) of ˜200 nM. Studies in CH103 VH+VL knock-in miceand Rhesus macaques using EnvSeq-1 have been completed and demonstrateproof of concept that sequential CH505 gp120s can initiate bnAb B cellclonal lineages in the setting of vaccination. The EnvSeq-1 vaccinebinds to CH103 precursors in CH103 bnAb knock-in mice and can expandthem with immunization in adjuvant. In Rhesus macaques, the gp120EnvSeq-1 vaccine can induce antibodies with the characteristics ofprecursors of CD4 binding site bnAbs. These characteristics includeantibodies that differentially bind CH505 Env but not Env with anisoleucine deletion at aa 371 that disrupts the CD4 binding site,antibodies that use similar VH4 and V13 genes to the human CH103 bnAb,and antibodies that neutralize the tier lb T/F variant CH505 4.3 as wellas some tier 2 viruses.

Utility of gp120s as Sequential Envs

Whether a native trimer is needed for this purpose or if a highlyantigenic Env subunit will suffice is yet unknown, but studies in micein basic B cell biology have demonstrated that what is important for Bcell survival in the germinal center (GC) is the optimal affinity of theimmunogen for the GC B cell receptor (BCR)^(13,14). A key question iswhether gp120 or gp140 trimers are preferred immunogens in a sequentialregimen. Emerging data have demonstrated that gp120s or their fragmentscan engage bnAb UCAs and expand CD4bs bnAb precursors^(5,15,16). Incontrast, recent data with soluble individual trimers have demonstratedthat they have only induced autologous tier 2 neutralizing antibodiesagainst glycan-bare spots and not bnAb epitopes^(17,18). Thus, it isappropriate at this time to continue to study gp120 immunogens in man totest the hypothesis that sequential immunogens can initiate bnAblineages. Whether boosting later in the immunization sequence with atrimeric Env will be needed will be tested in future studies.

The EnvSeq-2 Sequential Vaccine from CH505 is Designed to Induce CD4Mimicking-Type of CD4 Binding Site bnAbs

In this application we propose to extend the test of sequential Envimmunizations in man for initiation of broadly neutralizing antibodiesto test in a human Phase I clinical trial of a new series of CH505 Envs(the EnvSeq-2 vaccine) specifically designed to induce a broader andmore potent bnAb type, the CH235-like VH1-46 utilizing CD4 mimickingbroad neutralizing antibody with 90% breadth and 0.6 mcg/ml inhibitoryconcentration 50 (IC50).

Design of a Sequential Immunogen (EnvSeq-2) to Initiate VH1-46 CD4Mimicking CD4 Binding Site Antibody Lineages

Provided herein is a new set of immunogens based on the recent workdescribing the sequence of events that occurred with the development ofCD4 mimicking CD4 binding site bnAb lineage, CH235¹².

From this work, a natural mutant of the CH505 T/F Env called CH505.M5was found with one amino acid difference than the CH505 T/F strain,i.e., a single N279K change, that occurred very early on afterinfection; M5 binds to the CH235 UCA (˜0.5 micromolar)³. Thus, M5 is theinitiating Env for CD4 mimicking CD4 binding site antibodies in thecontext of the EnvSeq-2 vaccine.

Next, a set of 6 mutations at amino acids 97, 275, 278, 279, 281, and471 in the Env binding site to the CH235 lineage (FIG. 10 ), that wereassociated with escape from early CH235 antibody lineage members fromautologous CH505 viruses were identified and three additional Envschosen based on mutations at these sites.

TABLE 14 EnvSeq-2 vaccine and key amino acid mutations as well as V5length in vaccine Env gp120 components. Vaccine Env aa97 aa275 aa279aa281 aa471 Env V5 length CH505 M5 K E T V G 8 CH505 30.25 K E T A G 10CHO505 53.35 E E T G G 11 CH505 53.29 K E T A E 11

Importantly, the later CH235 antibody lineage members acquired theability to recognize viruses with these 6 Env mutations, presumably dueto the selection imposed by exposure to the resistance mutations invivo. These late Ch235 antibodies (such as the most potent CH235.12antibody) had expanded breadth due to selection for recognition of these6 mutations.

These chosen Envs in EnvSeq-2 vaccine are not associated with the bestbinding of the antibodies at intermediate steps as was done for designof the EnvSeq-1 vaccine above. Rather, as the increase in breadth at inthe heterologous panel coincided with a gained capacity to recognizeresistance mutations, Envs were selected based on their potential toexpand CH235 antibody lineage recognition in order to tolerate these 6key and common neutralization resistance-Env mutations. Nonetheless theselected Envs indeed had capacity to sequentially bind to lineagemembers (FIG. 20 ).

Finally, the fifth hypervariable loop (V5) region length was also astrong signature for recognition of CH505 viruses by CH235 antibodies,and early lineage members could only bind and neutralize short V5s.Longer V5s were selected by the early antibodies, and later antibodiescould recognize viruses with longer V5s, which are more representativeof the heterologous tier 2 HIV virus population. Thus, a final keycriterion for selection of sequential Envs in the EnvSeq-2 vaccine wasprogressive lengthening of V5 (Table 14). Thus, the EnvSeq-2 Envs areassociated with development of heterologous breadth from the CH235UCA→CH235→CH235.9→CH235.12.

The EnvSeq-2 set of immunogens are currently begin produced in non-GMPin pre-production runs, and during year 1 of the Staged VaccineContract, will be tested in vitro in recombinant protein immunizationsin both VH+VL humanized mice and rhesus macaques. In addition, a secondset of CH505 immunogens chosen based on affinity of binding to membersof the CH235 antibody lineage will be tested in similar immunizationstudies (a vaccine called EnvSeq-3, FIG. 21 ).

The optimal immunogen of the two sets of sequential Envs followingcomparison of EnvSeq-2 versus EnvSeq-3 will be chosen for GMP productionin preclinical studies based on the following criteria:

-   -   a) highest level of induction of memory B cell antibodies that        bind to CH505 Env and do not bind to CH505 Env with an        isoleucine deletion at amino acid position 371 that disrupts the        CD4 binding site (called “differential binding” memory B cells),    -   b) no neutralization of the tier 2 CH505 T/F virus (the CH235        UCA does not neutralize the CH505 tier 2 TF virus. However, if        the induced antibodies do neutralize the tier 2 TF CH505 virus,        then it will be an indication of immunogen driving a CH235        lineage further into lineage maturation).    -   c) highest level of neutralization of the tier 1b CH505 T/F        variant 4.3,    -   d) highest level of heterologous primary HIV strain neutralizing        antibodies induced.

In summary, provided are two selections of CH505 envelopes—FIG. 20(EnvSeq-2) or FIG. 21 (EnvSeq-3)—for use in immunization regimens. Insome embodiments these are used as recombinant CH505 Env gp120s(including gp120 delta N)), to be used in sequence following theadministration of the CH505 M5 priming Env that binds to the CH235 UCA.In other embodiments these are used in any other suitable form, forexample but not limited to stable SOSIP trimer designs, gp145s, gp140s,both cleaved and uncleaved, gp140 Envs with the deletion of the cleavage(C) site, fusion (F) and immunodominant (I) region in gp41—named asgp140ΔCFI (g140CFI), gp140 Envs with the deletion of only the cleavage(C) site and fusion (F) domain—named as gp140ΔCF (gp140CF), gp140 Envswith the deletion of only the cleavage (C)—named gp140ΔC (gp140C) (Seee.g. Liao et al. Virology 2006, 353, 268-282), gp150s, gp41s, which arereadily derived from the nucleic acid and amino acid gp160 sequences.

References for Example 6a

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Example 6B: Vaccine Antigen Design Based on the Evolution of Breadth ofthe CH235 bNAb Lineage

Four CH505 Vaccine Candidates Based on the Evolution of Breadth of theCH235 Lineage, Targeting the CD4bs

The mutant called CH505.M5 is the starting point for identifying CH505vaccine candidates. CH505.M5 is one amino acid different than the CH505TF strain, with a single N279K change, that occurred very early andconferred resistance to the cooperating CH103 lineages.

Identification of Signature Sites in the Contact Surface of the Antibody(<8.5 A)

Mutational patterns in the signature sites in the contact surface of theantibody are determined (in the global Tier II panel, as well as in oursubjects). These sites are related to heterologous and autologousneutralization sensitivity/resistance signatures. The pattern ofcritical interest is the set of mutations (in this case, 6 positionswith mutations that are common in the circulating population) that wereassociated with a high degree of resistance in the heterologouspopulation to early CH235 lineage members, but that were lessrestrictive for late lineage members. These amino acids were alsoassociated with a high degree of resistance to early antibodies amongCH505's Envs, and so escape in the autologous population. Later lineagemembers acquired the ability to recognize these mutations, presumablydue to the selection imposed by exposure to the resistance mutations invivo. These late antibodies then had expanded breadth at the populationlevel, presumably due to selection for recognition of these mutations.

These amino acids are not associated with the best binding of theantibodies at intermediate steps (earlier hypotheses for selecting Envswas to simply pick those that bound best to intermediate linagemembers). As the increase in breadth in the heterologous panel coincideswith a gained capacity to recognize resistance mutations, Envs arepicked based on their potential to expand Ab recognition to toleratecommon resistance mutations and also to require Envs that had at leastsome capacity to bind to lineage members, but placing emphasis oncovering common signatures, not on highest binders.

Hypervariable V5 region length was also a strong signature forrecognition, and early lineage members could only see short V5's. LongerV5s were selected by the early antibodies, and later antibodies couldrecognize viruses with longer V5s, which are more representative of theheterologous population.

The mutations conferring viral escape (or relative resistance) fromearly lineage antibodies are educating the later antibodies.

Later antibodies in the lineage gain breadth at the population levelbecause they evolved the capacity to recognize particular resistanceconferring amino acids that arose in vivo.

Envs that are associated with jump in breadth from theUCA→CH235→CH235.9→CH235.12 are defined. The amino acids that arestatistically most closely associated with distinct increases inbreadth, the heterologous signatures, are identified. These signaturesare related back to cycles of escape/recognition in vivo—exposure tothese signature amino acids seems to trigger the increase breadth. FIG.35 shows the heterologous panel heatmap of IC50s for CH235UCA, CH235,CH235.9, CH235.12, and VRC01 for 202 Envs.

Mutations that are common in the circulating population and areheterologous signatures are shown on the right of FIG. 36 . The 207 Mgroup panel is grouped by bNAb sensitivity—antibodies marked with anasterisk are sensitive. Selected based only on CH235 lineage signaturesat the population level, the env mutations in subject CH505 are shown inthe left of FIG. 36 . These contact signature amino acids are enrichedamong viruses that are resistance to CH235 and CH235.9, and sensitive toCH235.12.

Envs from CH505 that carried the signature mutations were picked,requiring at least some binding of later antibodies to the antigens andthat they carried modest increases in V5 length relative to M5 (FIG. 38). M5 was the best trigger for CH235 like antibodies. Env30.25 gave agentle nudge towards the most common mutations at the population level,where CH505 TF differed from consensus. An increase in V5 length ispresent. Env53.25 increase the V5 length, and adds three otherrelatively common mutations. Env 53.29 adds 471E, that may inhibitCH235.9 binding, but CH235.12 can recognized viruses with 471E. None ofthe CH235 Envs tested with the E275K mutation bound any of the CH235lineages, they were not included in the set. As there is no bindingdata, T278S comes up too late be included in the set.

Although CH235.12 binds Envs that carry K97E and G471E with lowaffinity, the differential capacity to recognize heterologous Envsbetween CH235.9 and CH235.12 is very strongly associated with CH235.12'sability to recognize Envs that have an E in either one of those 2positions, so including them here may enable selection of antibodiesthat can recognize these quite common mutations at the population level,that restrict CH235's early lineage member's breadth (FIG. 39 ).

A main difference between the choice of CH505 immunogens in FIG. 40(Selection F) and Selection G is in signature position 97 and 471. Theseare invariant among these six strains, with K97 and G471. But each hasan E among the antigens selected on FIG. 38 .

Example 7: Animal Studies

The immunogens of the invention, for example Selection F (M5, M11,20.14, 30.20, 30.12, 136.B18) could be tested in any suitable non-humananimal model. Immune responses, including B cell and T cell responses tothe vaccine, could be measured by any suitable assay and criteria, suchas but non limited plasma neutralization, plasma binding to vaccineand/or heterologous envelopes and/or viruses could be measured. Animalsstudies with various forms of the selected immunogens are contemplated:gp160 mRNA of M5, M11, 20.14, 30.20, 30.12, 136.B18 (NHP #141), 6-valentM5, M11, 20.14, 30.20, 30.12, 136.B18 as SOSIP trimers (NHP #142), mRNAof 6-valent stabilized SOSIP trimers of M5, M11, 20.14, 30.20, 30.12,136.B18 (NHP #140), gp145DNA of CH505M5 and CH505M11 as a prime and asubsequence boost(s), followed by 6-valent M5, M11, 20.14, 30.20, 30.12,136.B18 SOSIP trimers (e.g. NHP #139). In some embodiments the SOSIPtrimer is SOSIP v4.1. Any other trimer design is contemplated. Anysuitable adjuvant could be used. Studies could be performed in anysuitable animal model. Studies could be performed in adults andneonates.

TABLE 15 NHP Study #139: gp145 DNA M5 + M11(×2) + 6-valent 4.1 SOSIP inneonates. Samples Done at Bleed Receive @ Qty/Volume receiving DateBIDMC Instructions (I.D. Immunizations) Needed laboratory Wk 0 Wk 0Bleed + Immunize neonates only: EDTA+ + Freeze **Prebleed M5 gp 145 DNA(2 mg) + Stool + Rectal plasma in Processed M11 gp 145 DNA (2 mg)swabs + saliva + 250 uL at Bioqual IM IDEXX serum aliquots chems + CBC*Bioqual to freeze Wk 0 samples Wk 2 Wk2 Bleed all animals EDTA+ +Freeze Stool + Rectal plasma in swabs + saliva + 250 uL IDEXX serumaliquots chems + CBC Wk 4 Wk 4 Immunize neonates only: Stool + Rectal M5gp 145 DNA (2 mg) + swabs + saliva M11 gp 145 DNA (2 mg) IM Wk 6 Wk 6Bleed all animals EDTA+ + Freeze Stool + Rectal plasma in swabs +saliva + 250 uL IDEXX serum aliquots; all chems + CBC PBMCs Wk 8 Wk 8Immunize neonates only: Stool + Rectal M5 SOSIP 4.1 stable swabs +saliva trimer (25 ug) + M11 SOSIP 4.1 stable trimer (25 ug) In Poly ICLC(Hiltonol) = 200 ug IM Wk 10 Wk 10 Bleed all animals EDTA+ + FreezeStool + Rectal plasma in swabs + saliva + 250 uL IDEXX serum aliquots;all chems + CBC PBMCs Wk 12 Wk 12 Immunize neonates only: Stool + Rectal20.14 SOSIP 4.1 stable trimer swabs + saliva (50 ug) In Poly ICLC(Hiltonol) = 200 ug IM Wk 14 Wk 14 Bleed all animals EDTA + SST + Freezeserum Stool + Rectal and plasma swabs + saliva + in 250 uL IDEXX serumaliquots; all chems + CBC PBMCs Wk 16 Wk 16 Immunize neonates only:Stool + Rectal 30.20 SOSIP 4.1 stable trimer swabs + saliva (50 ug) InPoly ICLC (Hiltonol) = 200 ug IM Wk 18 Wk 18 Bleed all animals EDTA +SST + Freeze serum Stool + Rectal and plasma swabs + saliva + in 250 uLIDEXX serum aliquots; all chems + CBC PBMCs Wk 20 Wk 20 Immunizeneonates only: Stool + Rectal 30.12 SOSIP 4.1 stable trimer swabs +saliva In Poly ICLC (Hiltonol) = 200 ug IM Wk 22 Wk 22 Bleed all animalsEDTA + SST + Freeze serum Stool + Rectal and plasma swabs + saliva + in250 uL IDEXX serum aliquots chems + CBC Wk 24 Wk 24 Immunize neonatesonly: Stool + Rectal 136.B8 SOSIP 4.1 stable trimer swabs + saliva InPoly ICLC (Hiltonol) = 200 ug IM Wk 26 Wk 26 Bleed all animals EDTA +SST + Freeze serum Stool + Rectal and plasma swabs + saliva + in 250 uLIDEXX serum aliquots chems + CBC

TABLE 16 NHP study #140: mRNA 6-valent chimeric stabilized 4.1 trimersBleed Receive @ Samples Done at Date BIDMC Instructions (I.D.Immunizations) Qty/Volume Needed BIDMC Pre- Pre- Pre-LN biopsy(axillary) 6 ml EDTA + 2 ml Freeze bleed bleed Pre-Bleed all animalsSST + Plasma and Feb. 22, 2017 Feb. 23, 2017 NHP's: serum chemistryserum in 150796, 150798, 150794, 150252 (IDEXX) + CBC 250 uL (IDEXX)aliquots; all PBMC Wk 0 Wk 0 Bleed all animals and immunize: No samplingNo Feb. 28, 2017 Feb. 29, 2017 M5 chimeric stabilized trimer (kos)sampling mRNA-LNP 50 ug + M11 chimeric stabilized trimer (kos) mRNA-LNP50 ug ID = 10 sites on the back Give M5 and M11 separately at differentsites to avoid heterotrimers mRNA-LNPs: *diluted in calcium andmagnesium free PBS where needed. *once thawed are stored on ice andadministered within 2 hours NHP's: 150796, 150798, 150794, 150252 Wk 1Wk 1 Bleed all animals 6 ml EDTA + 2 ml Freeze Mar. 7, 2017 Mar. 8, 2017LN biopsy (inguinal) SST + Plasma and serum chemistry serum in (IDEXX) +CBC 250 uL (IDEXX) aliquots; all PBMC Wk 2 Wk 2 Bleed all animals 6 mlEDTA + 2 ml Freeze Mar. 14, 2017 Mar. 15, 2017 SST + serum and serumchemistry plasma in (IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs Wk4 Wk 4 Bleed all animals and immunize: 6 ml EDTA + 2 ml Freeze Mar. 29,2017 Mar. 30, 2017 20.14 chimeric stabilized trimer (kos) SST + serumand mRNA-LNP 50 ug serum chemistry plasma in ID = 10 sites on the back(IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs Wk 5 Wk 5 Bleed allanimals 6 ml EDTA + 2 ml Freeze Apr. 5, 2017 Apr. 6, 2017 LN biopsy(inguinal) SST + serum and serum chemistry plasma in (IDEXX) + CBC 250uL (IDEXX) aliquots; all PBMCs Wk 6 Wk 6 Bleed all animals 6 ml EDTA + 2ml Freeze Apr. 12, 2017 Apr. 13, 2017 SST + serum and serum chemistryplasma in (IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs Wk 8 Wk 8Bleed all animals and immunize: 6 ml EDTA + 2 ml Freeze Apr. 26, 2017Apr. 27, 2017 30.20 chimeric stabilized trimer (kos) SST + serum andmRNA-LNP 50 ng serum chemistry plasma in ID = 10 sites on the back(IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs Wk 9 Wk 9 Bleed allanimals 6 ml EDTA + 2 ml Freeze May 3, 2017 May 4, 2017 SST + serum andserum chemistry plasma in (IDEXX) + CBC 250 uL (IDEXX) aliquots; allPBMCs Wk 10 Wk 10 Bleed all animals 6 ml EDTA + 2 ml Freeze May 10, 2017May 11, 2017 SST + serum and serum chemistry plasma in (IDEXX) + CBC 250uL (IDEXX) aliquots; all PBMCs Wk 12 Wk 12 Bleed all animals andimmunize: 6 ml EDTA + 2 ml Freeze May 24, 2017 May 25, 2017 30.12chimeric stabilized trimer (kos) SST + serum and mRNA-LNP 50 ug serumchemistry plasma in ID = 10 sites on the back (IDEXX) + CBC 250 uL(IDEXX) aliquots; all PBMCs Wk 13 Wk 13 Bleed all animals 6 ml EDTA + 2ml Freeze May 31, 2017 Jun. 1, 2017 SST + serum and serum chemistryplasma in (IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs Wk 14 Wk 14Bleed all animals 6 ml EDTA + 2 ml Freeze Jun. 7, 2017 Jun. 8, 2017SST + serum and serum chemistry plasma in (IDEXX) + CBC 250 uL (IDEXX)aliquots; all PBMCs Wk 16 Wk 16 Bleed all animals and immunize: 6 mlEDTA + 2 ml Freeze Jun. 21, 2017 Jun. 22, 2017 136.B18 chimericstabilized trimer (kos) SST + serum and mRNA-LNP 50 ug serum chemistryplasma in ID = 10 sites on the back (IDEXX) + CBC 250 uL (IDEXX)aliquots; all PBMCs Wk 17 Wk 17 Bleed all animals 6 ml EDTA + 2 mlFreeze Jun. 28, 2017 Jun. 29, 2017 LN biopsy (axillary) SST + serum andserum chemistry plasma in (IDEXX) + CBC 250 uL (IDEXX) aliquots; allPBMCs Wk 18 Wk 18 Bleed all animals 6 ml EDTA + 2 ml Freeze Jul. 5, 2017Jul. 6, 2017 SST + serum and serum chemistry plasma in (IDEXX) + CBC 250uL (IDEXX) aliquots; all PBMCs Wk 20 Wk 20 Bleed all animals 6 ml EDTA +2 ml Freeze Jul. 19, 2017 Jul. 20, 2017 SST + serum and serum chemistryplasma in (IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs Wk 24 Wk 24Bleed all animals 6 ml EDTA + 2 ml Freeze Aug. 2, 2017 Aug. 3, 2017SST + serum and serum chemistry plasma in (IDEXX) + CBC 250 uL (IDEXX)aliquots; all PBMCs Wk 28 Wk 28 Bleed all animals 6 ml EDTA + 2 mlFreeze Aug. 16, 2017 Aug. 17, 2017 SST + serum and serum chemistryplasma in (IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs

In any of the methods of the invention, the mRNA immunogens aredelivered by a lipid nanoparticle (LNP) technology. The LNPs comprisesfour different lipids that could self assemble to 80-100 nm sizeparticles.

TABLE 17 NHP Study #141: mRNA 6-valent gp 160 membrane bound trimersReceive Samples Done at Bleed @ Qty/Volume receiving Date BIDMCInstructions (I.D. Immunizations) Needed laboratory Wk 0 Wk 0 Pre-LNbiopsy (axillary) Pre-LN biopsy Freeze serum Nov. 9, 2016 Nov. 10, 2016Immunize all 4 animals: (axillary and plasma 4 NHPs (4 NOTchallenged 4ml EDTA + 2 ml in 250 uL monkeys from #129 vaccinated): SST aliquots6858(M), 150250(F), 150793(M), 150795(M) M5 gp 160 membrane bound trimermRNA-LNP 50 ug + M11 gp 160 membrane bound trimer mRNA-LNP 50 ug ID = 60ul × 10 sites on the back Give M5 and M11 separately at different sitesto avoid heterotrimers mRNA-LNPs: *diluted in calcium and magnesium freePBS where needed. *once thawed are stored on ice and administered within2 hours Wk 1 Wk 1 Bleed all animals + Draining lymph 2 ml EDTA + FreezeNov. 16, 2016 Nov. 17, 2016 node biopsies (axillary) Draining lymphplasma in node biopsies 250 uL (axillary) aliquots Wk 2 Wk 2 Bleed allanimals 6 ml EDTA + 2 ml Freeze serum Nov. 21, 2016 Nov. 22, 2016 SSTand plasma in 250 uL aliquots; all PBMCs Wk 4 Wk 4 Bleed all animals andimmunize: 4 ml EDTA + 2 ml Freeze serum Dec. 6, 2016 Dec. 7, 2016 20.14gp 160 membrane bound SST and plasma trimer mRNA-LNP 50 ug in 250 uL ID= 60 ul × 10 sites on the back aliquots Wk 5 Wk 5 Bleed all animals +Draining lymph 3 ml EDTA + Freeze serum Dec. 13, 2016 Dec. 14, 2016 nodebiopsies (inguinal) Draining lymph and plasma node biopsies in 250 uL(inguinal) aliquots; all PBMCs Wk 6 Wk 6 Bleed all animals 3 ml EDTA + 2ml Freeze serum Dec. 20, 2016 Dec. 21, 2016 SST and plasma in 250 uLaliquots; all PBMCs Wk 7 Wk 7 Bleed all animals 3 ml EDTA Freeze Dec.27, 2016 Dec. 28, 2016 plasma in 250 uL aliquots; all PBMCs Wk 8 Wk 8Bleed all animals and immunize: 4 ml EDTA + 2 ml Freeze serum Jan. 4,2017 Jan. 5, 2017 30.20 gp 160 membrane bound SST and plasma trimermRNA-LNP 50 ug in 250 uL ID = 60 ul × 10 sites on the back aliquots Wk 9Wk 9 Bleed all animals 3 ml EDTA + 2 ml Freeze serum Jan. 11, 2017 Jan.12, 2017 SST and plasma in 250 uL aliquots Wk 10 Wk 10 Bleed all animals3 ml EDTA + 2 ml Freeze serum Jan. 18, 2017 Jan. 19, 2017 SST and plasmain 250 uL aliquots; all PBMCs Wk 12 Wk 12 Bleed all animals andimmunize: 4 ml EDTA + 2 ml Freeze serum Jan. 31, 2017 Feb. 1, 2017 30.12gp 160 membrane bound SST and plasma trimer mRNA-LNP 50 ug in 250 uL ID= 60 ul × 10 sites on the back aliquots Wk 13 Wk 13 Bleed all animals 3ml EDTA + 2 ml Freeze serum Feb. 8, 2017 Feb. 9, 2017 SST and plasma in250 uL aliquots; all PBMCs Wk 14 Wk 14 Bleed all animals 3 ml EDTA + 2ml Freeze serum Feb. 14, 2017 Feb. 15, 2017 SST and plasma in 250 uLaliquots; all PBMCs Wk 16 Wk 16 Bleed all animals and immunize: 4 mlEDTA + 2 ml Freeze serum Feb. 28, 2017 Mar. 1, 2017 136.B18 gp 160membrane bound SST and plasma trimer mRNA-LNP 50 ug in 250 uL ID = 60 ul× 10 sites on the back aliquots Wk 17 Wk 17 Bleed all animals + Draininglymph 3 ml EDTA + 1 ml Freeze serum Mar. 7, 2017 Mar. 8, 2017 nodebiopsies (inguinal) SST + Draining and plasma lymph node in 250 uLbiopsies (inguinal) aliquots; all PBMCs Wk 18 Wk 18 Bleed all animals 3ml EDTA + 2 ml Freeze serum Mar. 14, 2017 Mar. 15, 2017 SST and plasmain 250 uL aliquots; all PBMCs Wk 20 Wk 20 Bleed all animals 4 ml EDTA +2 ml Freeze serum Mar. 28, 2017 Mar. 29, 2017 SST and plasma in 250 uLaliquots Wk 24 Wk 24 Bleed all animals 4 ml EDTA + 2 ml Freeze serumApr. 25, 2017 Apr. 26, 2017 SST and plasma in 250 uL aliquots Wk 28 Wk28 Bleed all animals 4 ml EDTA + 2 ml Freeze serum May 23, 2017 May 24,2017 SST and plasma in 250 uL aliquots

TABLE 18 NHP Study #142: 6-valent chimeric stabilized trimer protein(kos) Receive Samples Bleed @ Qty/Volume Done at Date BIDMC Instructions(I.D. Immunizations) Needed BIDMC Feb. 22, 2017 Feb. 23, 2017 Pre-LNbiopsy (axillary) 6 ml EDTA + 2 ml Freeze Pre-Bleed all animals SST +serum and NHP’s: serum chemistry plasma in 150251,6857, T244,T245(IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs Wk 0 Wk 0 Bleed allanimals and immunize: 6 ml EDTA + 2 ml Freeze Feb. 28, 2017 Feb. 29,2017 NHP#: SST + serum and 150251, 6857, T244, T245 serum chemistryplasma in M5 chimeric stabilized trimer (kos) + (IDEXX) + CBC 250 uL Milchimeric stabilizedtrimer (kos) (IDEXX) aliquots; all IM injectionsPBMCs Wk 1 Wk 1 Bleed all animals 6 ml EDTA + 2 ml Freeze Mar. 7, 2017Mar. 8, 2017 LN biopsy (inguinal) SST + serum and serum chemistry plasmain (IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs Wk 2 Wk 2 Bleed allanimals 6 ml EDTA + 2 ml Freeze Mar. 14, 2017 Mar. 15, 2017 SST + serumand serum chemistry plasma in (IDEXX) + CBC 250 uL (IDEXX) aliquots; allPBMCs Wk 4 Wk 4 Bleed all animals and immunize: 6 ml EDTA + 2 ml FreezeMar. 29, 2017 Mar. 30, 2017 20.14 chimeric stabilized trimer (kos) SST +serum and serum chemistry plasma in (IDEXX) + CBC 250 uL (IDEXX)aliquots; all PBMCs Wk 5 Wk 5 Bleed all animals 6 ml EDTA + 2 ml FreezeApr. 5, 2017 Apr. 6, 2017 LN biopsy (inguinal) SST + serum and serumchemistry plasma in (IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs Wk6 Wk 6 Bleed all animals 6 ml EDTA + 2 ml Freeze Apr. 12, 2017 Apr. 13,2017 SST + serum and serum chemistry plasma in (IDEXX) + CBC 250 uL(IDEXX) aliquots; all PBMCs Wk 8 Wk 8 Bleed all animals and immunize: 6ml EDTA + 2 ml Freeze Apr. 26, 2017 Apr. 27, 2017 30.20 chimericstabilized trimer (kos) SST + serum and serum chemistry plasma in(IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs Wk 9 Wk 9 Bleed allanimals 6 ml EDTA + 2 ml Freeze May 3, 2017 May 4, 2017 SST + serum andserum chemistry plasma in (IDEXX) + CBC 250 uL (IDEXX) aliquots; allPBMCs Wk 10 Wk 10 Bleed all animals 6 ml EDTA + 2 ml Freeze May 10, 2017May 11, 2017 SST + serum and serum chemistry plasma in (IDEXX) + CBC 250uL (IDEXX)) aliquots; all PBMCs Wk 12 Wk 12 Bleed all animals andimmunize: 6 ml EDTA + 2 ml Freeze May 24, 2017 May 25, 2017 30.12chimeric stabilized trimer (kos) SST + serum and serum chemistry plasmain (IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs Wk 13 Wk 13 Bleedall animals 6 ml EDTA + 2 ml Freeze May 31, 2017 Jun. 1, 2017 SST +serum and serum chemistry plasma in (IDEXX) + CBC 250 uL (IDEXX)aliquots; all PBMCs Wk 14 Wk 14 Bleed all animals 6 ml EDTA + 2 mlFreeze Jun.7, 2017 Jun.8, 2017 SST + serum and serum chemistry plasma in(IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs Wk 16 Wk 16 Bleed allanimals and immunize: 6 ml EDTA + 2 ml Freeze Jun.21, 2017 Jun.22, 2017136.B18 chimeric stabilized trimer SST + serum and (kos) serum chemistryplasma in (IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs Wk 17 Wk 17Bleed all animals 6 ml EDTA + 2 ml Freeze Jun.28, 2017 Jun.29, 2017 LNbiopsy (axillary) SST + serum and serum chemistry plasma in (IDEXX) +CBC 250 uL (IDEXX) aliquots; all PBMCs Wk 18 Wk 18 Bleed all animals 6ml EDTA + 2 ml Freeze Jul. 5, 2017 Jul. 6, 2017 SST + serum and serumchemistry plasma in (IDEXX) + CBC 250 uL (IDEXX) aliquots; all PBMCs

This protocol describes NHP immunization study with M5, M11, 20.14,30.20, 30.12, 136.B18 envelopes and SIVGag. In some embodiments thebelow vaccination regimen could be carried out with the proteinsdelivered as trimers, for example but not limited to SOSIP.III trimers.

TABLE 19 Samples Qty/Volume Bleed Date Instructions Needed Notes Pre(−12 to Collect pre samples Plasma, PBMC, serum, −4 weeks) (−12 to −4weeks) saliva, rectal swab, vaginal swab, LN (axillary) Wk 0 Vaccination#1: M5 + M11 Plasma, PBMC, serum, (EP1) Vaccine HIV env gp 145 DNA & SIVgag saliva, rectal swab, vaginal DNA (Conserved element CE prime swab,fecal sample followed by C0-delivery of CE & complete gag boost) DNAdose = 2 mg of each construct Protein HIV gp 120. Env dose = 200 ug ofeach protein Adjuvant = GLA-SE 25 ug Route: IM/EP Innovio (n = 5) Group1A Group 1B Group 1C Group 1D DNA + Protein co-immunization (both sides)= into same muscle Group 2A Group 2B Group 2C Group 2D DNA (Left side) +Protein (Right side) = separate sides and muscles Group 3A Group 3B ShamDNA and adjuvant co-immunization (Both Sides) = same muscle Group 4AGroup 4B Treatment naive Wk 1 LN G3A, G3B, G4A, G4B @ Lt only Plasma,PBMC, serum (EP1wk 1) Wk 2 Plasma, PBMC, serum, (EP1 Wk 2) saliva,rectal swab, vaginal swab, fecal sample Wk 8 Vaccination #2: 20.14Plasma, PBMC, serum (EP2) DNA dose = 2 mg of each construct Env dose =200 ug of each protein Wk 9 LN ing G1A, G2A @ Rt & Lt Plasma, PBMC,serum (EP2 wk 1) LN G3A, G3B, G4A, G4B @ Rt only Wk 10 BM G1A, G2APlasma, PBMC, serum, (EP2 wk 2) saliva, rectal swab, vaginal swab, fecalsample, vaginal bx, rectal bx Wk 16 Plasma, PBMC, serum Wk 24Vaccination #3: 30.20 Plasma, PBMC, serum (EP3) DNA dose = 2 mg of eachconstruct Env dose = 200 ug of each protein Wk 25 LN ing G1B, G2B Rt &Lt Plasma, PBMC, serum, (EP3 wk 1) Wk 26 BM G1B, G2B Plasma, PBMC,serum, (EP3 wk 2) saliva, rectal swab, vaginal swab, fecal sample Wk 32Plasma, PBMC Wk 40 Vaccination #4: 30.12 Plasma, PBMC, serum (EP 4) DNAdose = 2 mg of each construct Env dose = 200 ug of each protein Wk 41 LNing G1C, G2C Rt & Lt Plasma, PBMC, serum (EP 4 wk 1) Wk 42 BM G1C,G2CPlasma, PBMC, serum, (EP4 wk 2) saliva, rectal swab, vaginal swab, fecalsample, vaginal bx, rectal bx Wk 48 Plasma, PBMC Wk 56 Vaccination #5:136.B18 Plasma, PBMC, serum (EP5) DNA dose = 2 mg of each construct Envdose = 200 ug of each protein Wk 57 LN ing G1D, G2D Rt & Lt Plasma,PBMC, serum (EP5 wk 1) Wk 58 BM G1D, G2D Plasma, PBMC, serum, (EP5 wk 2)*Necropsy 2 animals saliva, rectal swab, vaginal swab, fecal sample Wk64 Plasma, PBMC Wk 74 Plasma, PBMC, serum, saliva, rectal swab, vaginalswab, fecal sample

Non-limiting example of an immunization protocols with Selection F (M5,M11, 20.14, 30.20, 30.12, 136.B18). In this example the immunogens aredelivered as mRNA formulated in nanoparticles. In some embodiments thestabilized trimers are of the design SOSIP.III.

Materials needed: Formulate mRNA for 6 monkeys. 6 doses×50 ug/nhp=300 ugof each mRNA construct.

Collections of Plasma, Serum, and PBMC: Collect all plasma and serum in250 uL aliquots and save all PBMCs. CBC collection: 850 uL from eachanimal

Animal studies using the above protocols could be carried out with theimmunogens of Selection G (EnvSeq-2), or Selection H (EnvSeq-3).

Animal studies with envelopes CH505 T/F, as stable trimers are alsocontemplated. Non-limiting examples of such studies include: CH505 T/Fas gp145 nucleic acid prime (once or twice), followed by sequentialSOSIP 4.1 trimers of CH505 T/F, CH505 w53.16, CH505 w78.33, CH505w100.B6. In some embodiments there is no nucleic acid prime andimmunization regimen comprises sequential SOSIP 4.1 trimers of CH505T/F, CH505 w53.16, CH505 w78.33, CH505 w100.B6. In some embodiments thenucleic acid is mRNA. In some embodiments the nucleic acid is DNA. Insome embodiments the DNA is administered via electroporation. In someembodiments of these studies, animals could be boosted with CH505w136.B8.

Example 8: Maturation Pathway from Germline to Broad HIV-1 Neutralizerof a CD4-Mimic Antibody

Antibodies with ontogenies from V_(H)1-2 or V_(H)1-46-germline genesdominate the broadly neutralizing response against the CD4-binding site(CD4bs) on HIV-1. Here we define with longitudinal sampling fromtime-of-infection the development of a V_(H)1-46-derived antibodylineage that matured to neutralize 90% of HIV-1 isolates. Structures oflineage antibodies CH235 (week 41 from time-of-infection, 18% breadth),CH235.9 (week 152, 77%) and CH235.12 (week 323, 90%) demonstrated thematuring epitope to focus on the conformationally invariant portion ofthe CD4bs. Similarities between CH235 lineage and five unrelated CD4bslineages in epitope focusing, length-of-time to develop breadth, andextraordinary levels of somatic hypermutation suggested commonalities inmaturation among all CD4bs antibodies. Fortunately, the requiredCH235-lineage hypermutation appeared substantially guided by theintrinsic mutability of the V_(H)1-46 gene, which closely resembledV_(H)1-2. We integrated our CH235-lineage findings with a second broadlyneutralizing lineage and HIV-1 co-evolution to suggest a vaccinationstrategy for inducing both lineages. See Cell. 2016 Apr. 7;165(2):449-63. GenBank Accession numbers of the CH235UCA heavy and lightchains are KU570032.1 and KU570045.1

Accession Numbers

Coordinates and structure factors for CH235, CH235.9 and CH235.12 incomplex with HIV-1 gp120 have been deposited with the Protein Data Bank(PDB ID 5F9W, 5F9O and 5F96). Next-generation sequencing data have beendeposited with the NCBI Sequence Reads Archive (SRP067168). Antibodyheavy and light chains have been deposited with GenBank(KU570032-KU570053).

Antibodies Names Correlation: See supra.

Example 9: HIV-1 Envelope Trimers and Other Envelope Designs

This example shows that stabilized HIV-1 Env trimer immunogens showenhanced antigenicity for broadly neutralizing antibodies, and are notrecognized by non-neutralizing antibodies. See also FIGS. 22-25,59,61-74, 81-82 . The example also describes additional envelopemodifications and designs. In some embodiments these envelopes,including but not limited to trimers are further multimerized, and/orused as particulate, high-density array in liposomes or other particles,for example but not limited to nanoparticles. Any one of the envelopesof the invention could be designed and expressed as described herein.

A stabilized chimeric SOSIP.III design was used to generate 10 CH505trimers. The CH505 TF SOSIP.III bound the CH103 UCA. Binding affinity ofthe CH103 lineage to the CH505 TF SOSIP.III correlates withneutralization potency against CH505 TF virus. This design wasapplicable to diverse viruses from multiple clades.

These results indicate that the native trimer on virions could haveinitiated the CH103 lineage during natural infection. CH103 recognizesall three protomers on the Env trimer. The SOSIP.III mimicked the nativetrimer on the virion in that stronger binding to it correlated withneutralization potency for the CH103 lineage. The SOSIP.III designenables soluble mimics of the native trimer to be tested as sequentialimmunogens in CH505 B cell lineage design vaccination. These trimersenable our efforts to utilize B cell lineage design with trimericimmunogens.

Elicitation of neutralizing antibodies is one goal for antibody-basedvaccines. Neutralizing antibodies target the native trimeric HIV-1 Envon the surface virions. The trimeric HIV-1 envelope protein consists ofthree protomers each containing a gp120 and gp41 heterodimer. Recentimmunogen design efforts have generated soluble near-native mimics ofthe Env trimer that bind to neutralizing antibodies but notnon-neutralizing antibodies. The recapitulation of the native trimercould be a key component of vaccine induction of neutralizingantibodies. Neutralizing Abs target the native trimeric HIV-1 Env on thesurface of viruses (Poignard et al. J Virol. 2003 January; 77(1):353-65;Parren et al. J Virol. 1998 December; 72(12):10270-4; Yang et al. JVirol. 2006 November; 80(22):11404-8.). The HIV-1 Env protein consistsof three protomers of gp120 and gp41 heterodimers that are noncovalentlylinked together (Center et al. J Virol. 2002 August; 76(15):7863-7.).Soluble near-native trimers preferentially bind neutralizing antibodiesas opposed to non-neutralizing antibodies (Sanders et al. PLoS Pathog.2013 September; 9(9): e1003618).

Sequential Env vaccination has elicited broad neutralization in theplasma of one macaque (Example 5B). The overall goal of our project isto increase the frequency of vaccine induction of bnabs in the plasma ofprimates with sequential Env vaccination. We hypothesized thatvaccination with sequential immunogens that target bnAb B cell lineageand mimic native trimers will increase the frequency of broadlyneutralizing plasma antibodies. One goal is increase the frequency ofvaccine induction of bnAb in the plasma of primates by sequential Envvaccination. It is expected that vaccination with sequential immunogensthat target bnAb B cell lineages and mimic the native trimers on virionswill increase the frequency of broadly neutralizing plasma antibodies.

Previous work has shown that CH505 derived soluble trimers are hard toproduce. From a study published by Julien et al in 2015 (Proc Natl AcadSci USA. 2015 Sep. 22; 112(38): 11947-11952.) it was shown that whileCH505 produced comparable amounts of protein by transient transfection,only 5% of the CH505 protein formed trimer which 5 times lower than thegold standard viral strain BG505. Provided here are non-limitingembodiments of well-folded trimers for Env immunizations.

Near-native soluble trimers using the 6R.SOSIP.664 design are capable ofgenerating autologous tier 2 neutralizing plasma antibodies in theplasma (Sanders et al. 2015), which provides a starting point fordesigning immunogens to elicit broadly neutralizing antibodies. Whilethese trimers are preferentially antigenic for neutralizing antibodiesthey still possess the ability to expose the V3 loop, which generallyresults in strain-specific binding and neutralizing antibodies aftervaccination. Using the unliganded structure the BG505.6R.SOSIP.664 hasbeen stabilized by adding cysteines at position 201 and 433 to constrainthe conformational flexibility such that the V3 loop is maintainedunexposed (Kwon et al. Nat Struct Mol Biol. 2015 July; 22(7): 522-531.).

Immunogen design. Provided are engineered trimeric immunogens derivedfrom multiple viruses from CH505. We generated chimeric 6R.SOSIP.664,chimeric disulfide stabilized (DS) 6R.SOSIP.664 (Kwon et al Nat StructMol Biol. 2015 July; 22(7): 522-531.), chimeric 6R.SOSIP.664v4.1(DeTaeye et al. Cell. 2015 Dec. 17; 163(7):1702-15. doi:10.1016/j.cell.2015.11.056), and chimeric 6R.SOSIP.664v4.2 (DeTaeye etal. Cell. 2015 Dec. 17; 163(7):1702-15. doi:10.1016/j.cell.2015.11.056). The 6R.SOSIP.664 is the basis for all ofthese designs and is made as a chimera of C.CH0505 and A.BG505. Thegp120 of C.CH505 was fused with the BG505 inner domain gp120 sequencewithin the alpha helix 5 (α5) to result in the chimeric protein. Thechimeric gp120 is disulfide linked to the A.BG505 gp41 as outlined bySanders et al. (PLoS Pathog. 2013 September; 9(9): e1003618). Theseimmunogens were designed as chimeric proteins that possess the BG505gp41 connected to the CH505 gp120, since the BG505 strain isparticularly adept at forming well-folded, closed trimers (FIG. 22A).This envelope design retains the CH505 CD4 binding site that is targetedby the CH103 and CH235 broadly neutralizing antibody lineages that wereisolated from CH505.

FIGS. 22 and 23 show nucleic acid and amino acid and sequences ofvarious CH505 envelope trimer designs. FIG. 23B shows an annotatedsequence of the SOSIP.III design. Based on the various SOSIP designs,any other suitable envelope, for example but not limited to CH505envelopes as described in WO2014042669 can be designed.

Recombinant envelopes as trimers could be produced and purified by anysuitable method. For a non-limiting example of purification methods seeRinge R P, Yasmeen A, Ozorowski G, Go E P, Pritchard L K, Guttman M,Ketas T A, Cottrell C A, Wilson I A, Sanders R W, Cupo A, Crispin M, LeeK K, Desaire H, Ward A B, Klasse P J, Moore J P. 2015. Influences on thedesign and purification of soluble, recombinant native-like HIV-1envelope glycoprotein trimers. J Virol 89:12189-12210.doi:10.1128/JVI.01768-15.

Multimeric Envelopes

Presentation of antigens as particulates reduces the B cell receptoraffinity necessary for signal transduction and expansion (See Baptistaet al. EMBO J. 2000 Feb. 15; 19(4): 513-520). Displaying multiple copiesof the antigen on a particle provides an avidity effect that canovercome the low affinity between the antigen and B cell receptor. Theinitial B cell receptor specific for pathogens can be low affinity,which precludes vaccines from being able to stimulate and expand B cellsof interest. In particular, very few naïve B cells from which HIV-1broadly neutralizing antibodies arise can bind to soluble HIV-1Envelope. Provided are envelopes, including but not limited to trimersas particulate, high-density array on liposomes or other particles, forexample but not limited to nanoparticles. See e.g. He et al. NatureCommunications 7, Article number: 12041 (2016), doi:10.1038/ncomms12041;Bamrungsap et al. Nanomedicine, 2012, 7 (8), 1253-1271.

To improve the interaction between the naïve B cell receptor and CH505SOSIP trimer protein we created to two constructs that can be presentedon particles. The first construct was made by fusing HIV-1 Envelopetrimer CH505 to ferritin (See FIG. 24G). Ferritin protein self assemblesinto a small nanoparticle with three-fold axis of symmetry. At theseaxis CH505 envelope protein was fused. Therefore, the assembly of thethree-fold axis also clusters three HIV-1 envelope protomers together toform an envelope trimer. Each ferritin particle has 6 axes which equatesto 6 CH505 trimers being displayed per particle. See e.g. Sliepen et al.Retrovirology201512:82, DOI: 10.1186/s12977-015-0210-4; See also FIG.24H-J.

Another approach to multimerize expression constructs usesstaphylococcus Sortase A transpeptidase ligation to conjugate CH505envelope trimers to cholesterol. The CH505 trimers can then be embeddedinto liposomes via the conjugated cholesterol. To conjugate the CH505trimer to cholesterol either a C-teminal LPXTG tag (SEQ ID NO: 396) or aN-terminal pentaglycine repeat tag (SEQ ID NO: 307) was added to theCH505 envelope trimer gene. Cholesterol was also synthesized with thesetwo tags. Sortase A was then used to covalently bond the tagged CH505envelope to the cholesterol. The sortase A-tagged trimer protein canalso be used to conjugate the trimer to other peptides, proteins, orfluorescent labels.

The invention provides design of envelopes and trimer designs whereinthe envelope comprises a linker which permits addition of a lipid, suchas but not limited to cholesterol, via a Sortase A reaction. See e.g.Tsukiji, S. and Nagamune, T. (2009), Sortase-Mediated Ligation: A Giftfrom Gram-Positive Bacteria to Protein Engineering. ChemBioChem, 10:787-798. doi:10.1002/cbic.200800724; Proft, T. Sortase-mediated proteinligation: an emerging biotechnology tool for protein modification andimmobilisation. Biotechnol Lett (2010) 32: 1.doi:10.1007/s10529-009-0116-0; Lena Schmohl, Dirk Schwarzer,Sortase-mediated ligations for the site-specific modification ofproteins, Current Opinion in Chemical Biology, Volume 22, October 2014,Pages 122-128, ISSN 1367-5931, dx.doi.org/10.1016/j.cbpa.2014.09.020;Tabata et al. Anticancer Res. 2015 August; 35(8):4411-7.

The lipid modified envelopes and trimers could be formulated asliposomes. Any suitable liposome composition is contemplated.

Non-limiting embodiments of envelope designs for use in Sortase Areaction are shown in FIG. 24 B-D.

Design of Trimers with Readthrough Codons

The development of clonal cell lines that highly express trimeric HIV-1Envelope will facilitate manufacturing of high quality proteins forclinical and research purposes. However, it is challenging to identifythe cells that express trimeric protein among the many cells makingvarious forms of HIV-1 Envelope with in the cell population. To identifycells expressing trimeric HIV-1 Envelope protein, we designed anexpression construct that simultaneously produces both secreted Envelopeprotein as well as membrane anchored Envelope protein. The secretedEnvelope protein can be purified using standard methods and results inunaltered soluble envelope. The membrane-anchored Envelope proteinserves to mark the cells within a population of cells that expressestrimeric Envelope. More specifically, the trimeric Envelope expressingcells are sorted by fluorescence-activated cell sorting using a HIV-1trimer specific antibody. The sorted cells can then be used to initiateclonal populations of cells that have been phenotypically shown toexpress the protein of interest.

The expression construct is designed by taking advantage of the amberstop codon UAG in messenger RNA. The codon UAG usually signifies the endof the polypeptide sequence, but at a low rate the ribosome canreadthrough this stop codon and continue to elongate the polypeptidechain. We incorporated this stop codon into our protein constructfollowed by the natural BG505 gp41 transmembrane and cytoplasmic tailsequence ended with two stop codons. Therefore, when the stop codon isreadthrough a membrane-anchored gp120/gp41 heterodimer is formed.Loughran et al. (2014) identified that the efficiency of readthroughcould be increased by flanking the amber stop codon with the nucleotidesCTA. Readthrough could be even further augmented with the addition ofCTAG nucleotides after the amber stop codon. We engineered expressionconstructs with both modifications to ensure an optimal ratio ofmembrane-anchored and secreted trimeric Envelope protein. Since the CTAGcreates a shift in reading frame we added GC nucleotides after the CTAGmotif to preserve the original reading frame. The addition of CTAGGCresults in the membrane anchored protein having a leucine and glycineresidue expressed before the transmembrane domain. FIG. 24A showsnon-limiting examples of readthrough designs. FIG. 24E and FIG. 24F showexpression of “CTA” and “CTAGGC” designs in transiently transfected 293Fcells. Any one of the envelopes of the invention could be designed andexpressed as readthrough envelopes.

Example 10: HIV Envelope Modifications for Germline Targeting of CD4bsBroadly Neutralizing Antibodies

Germline B cell stimulation is a key initial step in the ability of HIVvaccines to elicit broadly neutralizing antibodies (bNAbs). Several bNAblineages are known to target the CD4 binding site of HIV-1 envelopeglycoprotein gp120, and these lineages are of particular interest forvaccines. Here we describe specific modifications of HIV-1 gp120 andgp140 to trigger germline activation and drive subsequent B cellmaturation of CD4bs bNAbs. These modifications are two-fold: 1)site-specific mutagenesis of a glycine residue at position 458 of gp120,changing this residue to a tyrosine (G458Y mutation) and 2) biosynthesisof the G458Y mutated envelope glycoproteins in cells lacking the enzymeN-acetylglucosaminyltransferase, resulting in an enrichment of Man5glycoforms of N-linked glycans that would otherwise be processed intocomplex-type glycans. Together these modifications permit the envelopeglycoproteins of HIV-1 strain CH0505 to interact with germline forms ofthe CD4bs bNAb CH235.

We began these studies by testing the potency of CD4bs bNAbs and otherHIV-1 bNAbs against viruses that were produced in either 293T or293s/GnTI^(−/−) cells. The latter cells were used to produceMan5-enriched glycoforms of pseudoviruses, with the rationale that arelatively small Man5 glycan would replace larger complex-type glycansthat naturally exist and contribute to CD4bs masking. Theoligosaccharide composition of HIV-1 Env consists mostly ofunder-processed high mannose (Man5-9 GlcNac2) glycans because stericconstraints imposed by the highly glycosylated and trimeric structureEnv impede the actions of α-mannosidases that are needed for completeprocessing (1-4). The smaller fraction of fully processed glycans existsmainly as sialylated bi-, tri- and tetra-antennary complex-type glycans(5-7), some of which border the CD4bs (5, 8). Importantly, nascent Envglycans that are trimmed by α-mannosidases and progress to complex-typeglycans will remain as under-processed Man5 glycans in the absence ofthe enzymeUDP-N-acetylglucosamine:α-D-mannoside-β1,2-N-acetylglucosaminyltransferase(GnTI) (9), which is responsible for attachment of GlcNAc to Man5GlcNAc2in the medial-Golgi as a requisite step for complete processing. HIV-1Env proteins produced in 293s/GnTI^(−/−) cells are known to be enrichedfor Man5 glycans, although as expected under-processed high mannoseglycoforms (Man6-9) also exist (9, 10). There is at least one report ofimproved potency of mature CD4bs bNAbs against viruses produced inGnTI^(−/−) cells (11).

Shown in FIG. 75 are the neutralization potencies of a panel of HIV-1bNAbs to multiple epitopes. The bNAbs were assayed against a tier 2strain of HIV-1 Env-pseudotyped virus (TRO.11) produced in either 293Tor 293s/GnTI^(−/−) cells (Man5-enrichment). With the exception ofIgG1b12 and HJ16, the CDbs bNAbs showed markedly greater potency againstMan5-enriched virus. HJ16 was negatively impacted by Man5-enrichment,while IgG1b12 failed to neutralize both forms of the virus.Man5-enrichment had little or no impact on the neutralizing activity ofother bNAbs.

Despite the improved potency of many mature CD4bs bNAbs againstMan5-enriched virus, germline-reverted forms of these bNAbs possessed nodetectable neutralizing activity against either form of the virus (FIG.75 ). We next sought to determine whether neutralization by germlineforms of the CD4bs bNAbs would be detected by combining Man5-enrichmentand targeted glycan removal. Our initial efforts used targetedglycan-deleted Envs designed by others to bind germline-reverted formsof VRC01-class CD4bs bNAbs. We began by evaluating a series ofglycan-deleted variants of the clade C strain 426c described byStamatatos and colleagues (12, 13). A V1-V3 deleted form of 426c gp140lacking three glycans, one at position 276 in loop D that contacts thelight chains of VRC01 and NIH45-46 (14, 15), and two at positions 460and 463 in V5 that modulate VRC01 sensitivity (16), permit nanomolaraffinity binding of germline-reverted forms of VRC01 and NIH45-46,whereas binding is undetectable against wild-type 426c gp140 (12). Thesemutations also permit activation of B cells expressing germline-revertedBCRs of VRC01 and NIH45-46 in vitro (12), and they activategermline-reverted BCR of 3BNC60 in transgenic mice (13).

We examined parental 426c and three variants of this virus containing asingle mutation that removes the 276 glycan (426c.SM), a double mutationthat removes 460 and 463 glycans (426c.DM), or a triple mutation thatremoves all three glycans (426c.TM). To preserve infectivity, the V1-V3deletion that was introduced in the purified protein to facilitateexposure of the CD4bs remained intact in the Env-pseudotyped viruses.The neutralization phenotype of these viruses was extensivelycharacterized with HIV-1 sera, a panel of mAbs that preferentiallyneutralize Tier 1 viruses, and a panel of bNAbs (Table 20, leftcolumns). Loss of 1, 2 or 3 glycans had little or no effect on HIV-1sera and did not render the virus sensitive to mAbs that preferentiallyneutralize Tier 1 viruses. Thus the glycan-deleted viruses maintained aTier 2 phenotype. The viruses all resist neutralization by the MPER bNAb2F5, the V2-glycan bNAbs CH01, PG9, PG16 and PGDM1400, the V3-glycanbNAbs PGT121 and PGT128, the glycan bNAb 2G12 and the CD4bs bNAbs HJ16and b12. All four viruses were sensitive to the MPER bNAbs 4E10 andDH511.2_K3, the V3-glycan bNAb 10-1074 and the gp120/gp41 bNAbs PGT151and VRC34.1, and these bNAbs were not affected by glycan deletion.

The only bNAbs clearly affected by glycan deletion were the CD4bs bNAbsVRC01, 3BNC117, VRC-CH31 and CH103 (boxed in red in Table 20). Inparticular, VRC01 and 3BNC117 were approximately 10-100 times morepotent against 426c.TM than against the parental virus. Enhanced potencyof 3BNC117 required all 3 glycans to be removed. VRC01 also requiredremoval of all 3 glycans for maximum potency but unlike 3BNC117 itexhibited moderately enhanced potency seen against the single and doublemutants. VRC-CH31 exhibited potent activity against the parental virusand double mutant but was inactive against the single and triple mutant,indicating a strict requirement for the presence of the N276 glycan. ForCH103, modest neutralizing activity was seen against the double andtriple mutant, while the parental and single mutant resistedneutralization at the highest concentration tested (40 μg/ml). All fourviruses were resistant to CH235. Thus, overall the glycan-deletedvariants of 426c provided little or no advantage for detecting CH103 andCH235 when produced in 293T cells.

Despite a nearly 100-fold improved potency of VRC01 against 426c.TM, wewere unable to detect neutralization of this virus by germline-revertedVRC01. Part of the reason may due to the fact that the virus contains anintact V1-V3 region, whereas this region was deleted in the 426c.TMgp140 antigen that bound germline-reverted VRC01. Because V1-V3-deletedEnv-pseudotyped viruses are non-infectious, we sought to determinewhether Man5-enrichment would serve as an alternative strategy tofurther unmask the CD4bs on 426c.TM and enable detection of neutralizingactivity by germline-reverted VRC01.

When parental 426c Env and the single, double and triple glycan-deletedvariants of this Env were made as pseudoviruses in GnTI^(−/−) cells andassayed in TZM-bl cells, all four viruses were infectious and maintaineda Tier 2 neutralization phenotype (Table 20, columns on the right).Notably, they were also remarkably sensitive to neutralization byseveral CD4bs bNAbs (VRC01, 3BNC117, VRC-CH31 and CH103) compared totheir 293T-grown counterparts (Table 20 and FIG. 76 ). Man5-enriched426c.TM provided the most sensitive detection of VRC01, 3BNC117 andCH103 (IC50 of 0.015, 0.003 and 0.09 μg/ml, respectively), whereasMan5-enriched 426c.DM provided the most sensitive detection of VRC-CH31(0.02 μg/ml). Man5-enrichment had little or no measurable effect onbNAbs to most other epitopes. The only exception was a 100-folddiminished potency of the gp120/gp41 bNAb PGT151 against theMan5-enriched viruses. Man5-enrichment had no impact on anothergp120/gp41 bNAb, VRC34.1. These latter two bNAbs recognize overlappingbut distinct epitopes (17).

We tested whether the 426c glycan mutants produced in GnTI^(−/−) cellswould permit detection of neutralization by a germline-reverted form ofCD4bs bNAbs. As shown in FIG. 77 (top), near germline-reverted VRC01(containing mature HCDR3) neutralized Man5-enriched 426c.SM and 426c.TMwith IC50s of 0.9 μg/ml and 2.5 μg/ml, respectively. Little or noactivity was detected against Man5-enriched 426c.DM and 426c, indicatinga dependency on the presence of the N276 glycan, which is not the casefor mature VRC01. No activity was detected against any of the 426cviruses produced in 293T cells, indicating a requirement for bothMan5-enrichment and removal of targeted glycans to permitneutralization. Notably, no neutralization of the parental or targetedglycan-deleted viruses was seen with a more germline form of VRC01,whether the viruses were grown in 293T or GnTI^(−/−) cells (data notshown).

We tested near germline forms (mature HCDR3) of several additionalVRC01-class bNAbs (VRC03, VRC04, VRC07, VRC18b, VRC20, VRC23 andVRC-CH31) and found that Man5-enriched 426c.TM permits detection ofneutralization by near germline VRC07 (IC50=1.6 μg/ml) and VRC20(IC50=4.6 μg/ml) (FIG. 78A). We also tested four intermediates ofVRC-CH31. All intermediates of the VRC01-CH31 lineage neutralized 426cand 426c.DM produced in 293T cells; however, dramatic improvements inneutralization potency of 50-80 fold were seen against the Man5-enriched426c.DM (FIG. 77 , bottom).

We further showed that neutralization of Man5-enriched 426c.TM by neargermline VRC01-class bNAbs is completely abolished when a VRC01 escapemutation (D279K) (16) is introduced (FIG. 78B). These results indicatedthat we are able to detect near-germline forms of VRC01-class bNAbs andconfirm their epitope specificity.

While Man5-enriched glycoforms of glycan-deleted 426c Envs were usefulfor detecting near germline forms of VRC01-class bNAbs, they were notcapable of detecting neutralization by germline and early intermediatesof other CD4bs bNAb lineages, including CH103 and CH235. We investigatedtargeted glycan-deleted variants of the autologous transmitted/founderEnv (CH0505TF) that evolved and gave rise to CH103 and its CH235 helperlineage (18, 19). As seen in Table 21, CH0505TF lacking four glycans atpositions 197, 461/462, 276 and 362 (CH0505TF.gly4)demonstrated >1,000-fold enhanced sensitivity to early intermediates ofthe CH103 lineage compared to parental CH0505TF. Interestingly,Man5-enrichment showed only minor enhancement in sensitivity to theseintermediates, with the exception of germline CH103 that was onlydetected with Man5-enriched CH0505TF.gly4. Intermediates of CH235 weredetected at various levels with parental CH0505TF, CH0505TF.gly4 andCH0505TF.gly3.197 viruses, and this level of detection was much greaterwith Man5-enriched (GnTI^(−/−)) versions of the viruses. Noneutralization was detected with CH235 UCA.

We next sought to identify site-directed mutations that would allow usto map the epitopes of the activity detected with the CH103 and CH235intermediates. Although these epitopes are well-characterized, theidentification of diagnostic mutants would facilitate epitope mapping ofpolyclonal sera (e.g., vaccine sera) to determine whether positiveneutralizing activity is related to these lineages. We began by testingthe UCAs, intermediates and mature forms of CH235 for neutralizingactivity against a G458Y mutant of CH0505TF. This mutation was chosenbecause it is a known escape mutation for VRC01-class bNAbs (20).CH0505TF was chosen because it was the only virus neutralized by earlyintermediate CH235_I4_v2_4A (Table 21). Surprisingly, rather than renderthe viruses less sensitive to neutralization, the presence of thismutation rendered the virus more susceptible to neutralization by theCH235 lineage, an effect that was more pronounced with Man5-enrichedvirus (Table 22). Even more remarkable, the combination of G458Y andMan5-enrichment now permitted detection of neutralization by threeinferred UCAs of CH235 (Table 22, FIG. 79 ). No neutralization by theUCAs was detected when the G458Y mutant virus was produced in 293Tcells, or when the parental virus was produced in 293s GnTI^(−/−) cells,indicating a requirement for both the G458Y mutation andMan5-enrichment.

FIG. 80 show a crystal structure of CH235 antibody with a gp120envelope, and a model of the CH235 UCA interaction a gp120 envelope withG458 and Y458. This figure shows one possible structural rationale forimproved neutralization of the CH505 T/F G458Y mutant virus by the CH235UCA. The figure shows that in the CH235 mature antibody gp120 complex,W50 in the CH235 CDRH2 pairs with the G458. Amino acid W is large, G458is small. But for the CH235 UCA, it is I50 in the germline. The I50W isa mutation, and an extremely improbable one. I50 is much smaller than W.So in the CH235UCA/gp120 interaction, the pair is I50 (small) and G458(small) which means contacts are disrupted. The G458Y mutation in theenvelope acts to restore the pairing. So in the CH235UCA/gp120interaction, I50 (small) is paired with Y458 (large).

Non-limiting example of additional possible mutations at position 458are as follows:

-   -   G458F    -   G458W    -   G458M    -   G458Q    -   G458R (R is 2nd most frequent in LANL db)    -   G458K    -   G458H    -   G458N.

Without being bound by any specific theory, these mutations are expectedto improve contacts like TYR at 458. These amino acid changes areselected based on size to increase potential contacts to I50 in CH235UCA CDRH2.

Binding and/or affinity of antibodies to HIV-1 Env proteins G458MutGnTI^(−/−) cells produced, can be measured by any suitable method, forexample but not limited to ELISA, SPR, and the like. See Example 1 andExample 8.

Any suitable GnTI−/− cell line could be use. See Bussow, K. in CurrentOpinion in Structural Biology 2015, 32:81-90; Chang et al. Structure.2007 March; 15(3): 267-273. Glycosylation patterns of the envelopesproduced in GnTI^(−/−) cells could be determined by any suitable method.

References for Example 10

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Example 10

TABLE 20 Characterization of the neutralization properties of 426c,426c.SM (N276D), 426c.DM (N460D/N463D) and 426c.TM (N276D/N460D/N463D)produced in either 293T cells or 293s/GnTI^(−/−) cells(Man5-enrichment). ID50/IC50 in TZM-bl ID50/IC50 in TZM-bl ReagentEpitope 426c 426c.SM 426c.DM 426c.TM 426c/GnTI 426c.SM GnTI 426C.DM GnTI426c.TM GnTI CHAVI-0406 Polyclonal 20 20 20 86 172 201 230 338CHAVI-0060 Polyclonal 40 31 31 52 100 144 95 338 CHAVI-0642 Polyclonal55 68 51 129 282 339 169 519 CHAVI-0293 Polyclonal 20 20 20 113 26 45 42543 CHAVI-0585 Polyclonal 199 264 225 451 963 1544 1152 3011 GMT 45 4743 124 165 234 178 627 2219 V3 >25 >25 >25 >25 >25 >25 >25 >25 2557V3 >25 >25 >25 >25 >25 >25 >25 >25 3074 V3 >25 >25 >25 >25 >2523 >25 >25 3869 V3 >25 >25 >25 >25 >25 >25 >25 >25 447-52DV3 >25 >25 >25 >25 >25 >25 >25 >25 838-12DV3 >25 >25 >25 >25 >25 >25 >25 >25 830A V2 >25 >25 >25 >25 654-30DCD4bs >25 >25 >25 >25 >25 >25 >25 >25 1008-30DCD4bs >25 >25 >25 >25 >25 >25 >25 >25 1570DCD4bs >25 >25 >25 >25 >25 >25 >25 >25 729-30DCD4bs >25 >25 >25 >25 >25 >25 >25 >25 F105CD4bs >25 >25 >25 >25 >25 >25 >25 >25 HJ16CD4bs >25 >25 >25 >25 >25 >25 >25 >25 sCD4 CD4bs >25 >25 >25 20.2 >25 2423 23 2G12 glycan >25 >25 >25 >25 >25 >25 >25 >25 2F5MPER >25 >25 >25 >25 >25 >25 >25 >25 4E10 MPER 3.32 1.52 1.00 0.98 4 3.83.7 4 DH511.2_K3 MPER 0.8 0.77 1.50 1.06 0.85 0.87 0.75 1.2 CH01V2-gly >25 >25 >25 >25 >25 >25 >25 >25 PG9V2-gly >5 >5 >5 >5 >5 >5 >5 >5 PG16 V2-gly >5 >5 >5 >5 >5 >5 >5 >5PGDM1400 V2-gly >25 >25 >25 >25 >5 >5 >5 >5 PGT121 V3-gly >5 >5 >5 >52.5 4.2 3.4 3.4 PGT128 V3-gly >5 >5 >5 >5 4.3 >5 >5 >5 10-1074 V3-gly0.05 0.12 0.10 0.16 0.03 0.04 0.03 0.03 PGT151 gp120/gp41 0.01 0.01 0.010.01 1.6 1.9 2.2 2.5 VRC34.1 gp120/gp41 0.08 0.06 0.09 0.08 0.05 0.070.04 0.07 IgG1b12 CD4bs >25 >25 >25 >25 >25 >25 >25 >25 VRC01 CD4bs 2.200.39 0.41 0.03 0.19 0.04 0.04 0.015 3BNC117 CD4bs 0.20 0.24 0.13 0.010.02 0.01 0.006 0.003 VRC-CH31 CD4bs 0.62 >25 0.73 >25 0.04 0.7 0.02 1.6CH103 CD4bs >40 >40 6.1 5.2 5.3 2.2 0.63 0.09 DH235CD4bs >50 >50 >50 >50 >25 >25 >25 >25

Example 10

TABLE 21 Enhanced detection of neutralizing activity by earlyintermediates of the CH103 and CH235 lineages of CD4bs bNAbs. Glycanpositions deleted: CH0505TF.gly4 (197, 461/462, 276, 362);CH0505TF.gly3.197 (461/462, 276, 362); CH0505TF.gly3.276 (197, 461/462,362); CH0505TF.gly3.461 (197, 276, 362). Env-pseudotyped viruses wereproduced in either 293T cells or 293S/GnTI−/− cells (Man5-enrichment).IC50 (ug/ml) in TZM-bl cells CH0505TF/ CH0505TF/ CH0505TF.gly4/CH0505TF.gly4/ CH0505TF.gly3.197/ CH0505TF.gly3.197/ 293T GnTI^(−/−)293T GnTI^(−/−) 293T GnTI^(−/−) Reagent ID#5444 ID#7309 ID#7691 ID#7698ID#7693 ID#7701 VRC01 0.09 0.03 0.002 0.001 0.023 0.009 VRC01/ >50 >5026.4 >50 >50 >50 gHvgLv CH103_UCA1.1_4A >50 >50 >50 6.4 >50 >50CH103_UCAGrand5 >50 >50 >50 >50 >50 >50CH103_IA_9_4A >50 >50 >50 >50 >50 >50 CH103_IA_8_4A 25.2 0.52 0.0040.001 8.5 0.14 CH103_IA_7_4A 4.7 0.072 0.002 0.001 5.9 0.02CH103_IA_6_4A >50 5.5 0.001 0.001 >50 2.2 CH103_IA_5_4A 7.6 0.54 0.0010.002 2.8 0.48 CH103_IA_4_4A 3.2 0.15 0.002 0.001 0.97 0.035 CH103_4A2.6 0.67 0.01 0.005 1.6 0.19 (Mature)CH235UCA_LL >50 >50 >50 >50 >50 >50CH235UCAtk_v2_4A/ >50 >50 >50 >50 >50 >50 293i CH235_I4_v2_4A/ >501.4 >50 >50 >50 >50 293i CH235_I3_v2_4A 5.3 0.1 17.7 0.8 >50 0.018CH235VH_I1_v2_4A/ 0.85 0.05 0.83 0.24 1.8 0.022 293i CH235_4A 0.35 0.030.022 0.004 0.16 0.006 (mature)

Example 10

TABLE 22 Neutralization of CH0505TF by UCAs and early intermediates ofCH235 is dependent on G458Y and Man5-enrichment (GnTI^(-/-)). IC50(μg/ml) in TZM-bl CH0505TF CH0505TF.G458Y CH0505TF CH0505TF.G458YAntibody 2931 293T GnT^(-/-) GnTI^(-/-) CH235UCA_LL >50 >50 >50 0.03CH235UCAtK_v2_4A/293i >50 >50 >50 0.8 CH235UCALRLL_V3_4/293i >25 >25 >250.16 CH235_I4_v2_4A/293i >50 >50 1.4 0.08 CH235_I3_v2_4A 5.3 0.99 0.120.03 CH23SVH_I1_v2_4A/293i 0.85 0.14 0.1 <0.02 CH235_4A (mature) 0.350.2 <0.02 <0.02

For CH235 UCA Nomenclature see Example 13. Sequences of DifferentCH235UCAs are Referenced in Example 8 and Shown in FIGS. 59A and 59B.

Example 11

Envelope Modifications that Permit Neutralization of HIV-1 byGermline-Reverted Forms of Broadly Neutralizing Antibodies to the CD4Supersite

The ability to stimulate germline B cells that give rise to broadlyneutralizing antibodies (bNAbs) is a major goal for HIV-1 vaccinedevelopment. BNAbs that target the CD4-binding site (CD4bs) of HIV-1 andexhibit extraordinary potency and breadth of neutralization areparticularly attractive to elicit with vaccines. Glycans that border theCD4bs and impede the binding of germline-reverted forms of CD4bs bNAbsare potential barriers to naïve B cell receptor engagement. Targeteddeletion of a subset of these glycans by sequon mutation has permittedbinding but not neutralization, suggesting additional barriers exist. Weproduced HIV-1 in cells lacking the enzymeN-acetylglucosaminyltransferase (GnTI-) to enrich for Man5 glycoforms ofN-linked glycans that would otherwise be processed into complex-typeglycans. Our rationale was that small Man5 would replace largercomplex-type glycans to further reduce steric barriers to germline CD4bsbNAb binding without disrupting native Env conformation. Targetedglycan-deleted HIV-1 produced in GnTI-cells was infectious andsusceptible to potent neutralization by several germline-revertedVRC01-class bNAbs; neither glycan modification alone was sufficient forneutralization. Neutralization also was observed for germline-revertedand early intermediates of CH235/CH235.12 (VH1-46) and CH103 (VH4-59).Neutralization by germline-reverted CH235/CH235.12 required both Man5enrichment and mutation of G458 in the V5 region of gp120 withouttargeted glycan deletion. These findings advance our understanding ofthe restrictions imposed by glycans in the elicitation of CD4bs bNAbsand provide a conceptual framework for improved vaccine designs.

Summary

Induction of broadly neutralizing antibodies (bNAbs) is a high priorityfor HIV-1 vaccines. Although these antibodies are made in HIV-1-infectedindividuals, it has not been possible to induce them with currentvaccine immunogens. One reason for this is that the immunogens are notable to engage appropriate germline B cells to initiate the response.Here we show that glycans on the HIV-1 envelope can be modified in waysthat should allow the envelope to stimulate germline B cells that giverise to a class of bNAbs targeting the CD4-binding site (CD4bs) ofenvelope gp120. These modifications involve the removal of selectglycans, together with changes in the composition of other glycans, withthe aim of exposing the CD4bs in a native conformation. An additionalmodification involves a glycine to tyrosine mutation (G458Y) in theCD4bs of gp120, which does not alter glycan composition. Inferredgermline and early intermediates of certain CD4bs bNAbs exhibitedneutralizing activity only when targeted glycan removal, or the G458Ymutation, was combined with an enrichment of Man5 glycoforms on HIV-1Env-pseudotyped viruses. Our findings suggest that such modifications,and reverse-engineered versions of them, have potential to initiate andmature CD4bs bNAb responses.

Introduction

The CD4-binding site (CD4bs) of HIV-1 envelope glycoproteins (Env) isessential for virus entry [1] and is susceptible to some of the mostpotent broadly neutralizing antibodies (bNAbs) described to date,neutralizing up to 98% of circulating strains [2-10]. These bNAbs alsoprevent and SHIV infection in nonhuman primates [11-16] and producetransient reductions in plasma viremia in infected humans [17, 18] andmacaques [19, 20]. Such features make CD4bs bNAbs highly attractive forvaccine development. Unfortunately, although the human immune system isclearly capable of making these antibodies in the setting of chronicinfection, all efforts to elicit them with vaccines in non-humanprimates and humans have failed [21].

A major roadblock is the high levels of somatic hypermutation requiredto bind an epitope that is conformationally masked and stericallyoccluded by surrounding glycans [7, 9, 22, 23]. Mature CD4bs bNAbsresemble CD4 in their mode of binding and contact the CD4-binding loopwhile avoiding or accommodating potential clashes with loop D and thefifth variable (V5) regions of gp120, often contacting both of theselatter regions [2, 22, 24]. Few immunoglobulin gene families appear togive rise to CD4bs bNAbs, most notably VH1-2 and the closely relatedVH1-46, both of which are utilized by the most potent CD4bs bNAbs (e.g.,VRC01, 3BNC117, N6, CH235.12). Binding of these bNAbs is mediated by theheavy and light chains and is dominated by the heavy-chain secondcomplementarity determining region (CDRH2) when either VH1-2 or VH1-46are utilized [2, 5, 10]. Other CD4bs bNAbs (e.g., CH103, VRC13, VRC16and HJ16) make use of multiple additional VH gene families, and theirbinding involves a CDRH3-dominated mode of recognition [6, 10].

Part of the reason why current immunogens fail to induce these bNAbs isthat they do not bind germline-reverted forms of CD4bs bNAbs [7, 9, 22,25-29] and therefore are unlikely to engage cognate naïve B cellreceptors (BCRs). Weak germline binding has been detected againstautologous Envs but it is not clear that this weak binding will providean adequate stimulus to naïve B [30, 31].

Relationships between antibody structure and function are serving as abasis to reverse-engineer improved germline-targeting immunogens for theVRC01 class of CD4bs bNAbs. Notably, germline-reverted forms of thesebNAbs are less positively charged [32] and their CDRH3 might play a moredominant role [33] than the mature bNAbs; both of these features couldpotentially influence interactions with complex-type glycans. Germlinebinding has been detected by introducing Env mutations that selectivelyremove glycans in the vicinity of the CD4bs that are predicted to clashwith germline forms of the bNAbs. Targeted removal of three glycans fromclade C strain 426c gp140ΔV1-V3, one at N276 in loop D that contacts thelight chains of VRC01 and NIH45-46 [22, 34], and two at N460 and N463 inV5 that modulate VRC01 sensitivity [35], permit nanomolar affinitybinding of germline-reverted forms of VRC01 and NIH45-46 [27]. Thesemutations also permit activation of B cells expressing germline-revertedBCRs of VRC01 and NIH45-46 in vitro [27], and they activategermline-reverted BCR of 3BNC60 in transgenic mice [36]. Deletion ofglycan N276 is also one central design feature of engineered outerdomain, germline-targeting (eOD-GT) immunogens that bind germline formsof the VRC01 class of bNAbs and activate germline-reverted BCR inknock-in mice [26, 37, 38].

HIV-1 Env is one of the most heavily glycosylated proteins known, with aglycan content that accounts for approximately 50% of its molecular mass[39]. A majority of these glycans exist as under-processed Man5-9GlcNac2glycoforms owing to steric constrains imposed by the dense clustering ofglycans and the trimerization of gp120-gp41 heterodimers that impede theactions of α-mannosidases required for complex glycan formation [40-43].A predominance of high mannose glycans is seen with multiple forms ofEnv produced in different cell types [44-51], where a higher abundanceof Man5GlcNac2 is present on virions and membrane associated Env than onrecombinant gp120 and gp140 proteins [40, 44, 47]. The smallerproportion of fully processed glycans exists mainly as sialylated bi-,tri- and tetra-antennary complex-type glycans [4, 47, 52, 53], a portionof which surround the CD4bs [4, 54].

Complex-type glycans are arrested at Man5GlcNac2 in the absence of theenzyme N-acetylglucosaminyltransferase (GnTI) [55], which is responsiblefor attachment of GlcNAc to Man5GlcNAc2 in the medial-Golgi as arequisite step for complete processing. There is at least one report ofimproved neutralization potency of mature CD4bs bNAbs against Envsproduced in GnTI-cells [56]. Here we converted complex-type glycans intosmaller Man5GlcNac2 in the context of other Env modifications to reducesteric barriers to germline bNAbs without disrupting native Envconformation. We examined this by requiring neutralization ofEnv-pseudotyped viruses as proof germline bNAb engagement of nativefunctional Env.

Results

Enhanced neutralization potency of mature CD4bs bNAbs against Envsproduced in GnTI-cells

Multiple BNAbs were assessed for neutralizing activity againstEnv-pseudotyped viruses produced in either 293T or 293S GnTI-cells. Thelatter cells were used to generate Man5-enriched Env, with the rationalethat relatively small Man5 would replace larger complex-type glycansthat contribute to CD4bs masking. Initially, three mature CD4bs bNAbs(VRC01, 3BNC117 and VRC-CH31) were assayed against Envs from strainsCE1176 and WITO. Greater potency (often >10-fold) was seen againstGnTI-Envs for all three bNAbs (FIG. 84A). A third Env, TRO.11, wasassayed with a wider range of mature bNAbs covering multiple epitopes(FIG. 84B). With the exception of IgG1b12 and HJ16, the CD4bs bNAbsagain showed enhanced potency against GnTI-Env. HJ16 was less potentagainst GnTI-Env, while IgG1b12 was non-neutralizing. HJ16 requiresgp120 glycan N276 [57], and it is possible that occupation of this siteby Man5GlcNAc2 is not tolerated by HJ16. GnTI- had little or no impacton bNAbs to epitopes outside the CD4bs. Notably, no neutralization wasdetected with germline-reverted forms of CD4bs bNAbs (FIG. 84A).

Complementarity of Man5-enrichment and targeted glycan deletion forneutralization by mature CD4bs bNAbs

We next examined a combination of GnTI-production and targeted deletionof one or more glycans surrounding the CD4bs. Mutants of 426c Env wereused that lacked glycan N276 (426c.SM), two glycans at N460 and N463(426c.DM), or all three glycans (426c.TM) [27]. A fourth mutant,426c.TM4, lacked all three glycans except that glycan N276 was removedby introducing S278R [36]. TM4 also contained a G471S mutation thatfacilitates germline bNAb binding to eOD-GT6 [26].

The glycan-deleted Envs, whether produced in 293T or GnTI-cells,maintained a tier 2 neutralization phenotype with HIV-1 sera and weremostly resistant to mAbs that preferentially neutralize Tier 1 Envs(non-neutralizing Abs) (Table 24). Envs produced in GnTI-cells were moresensitive to HIV-1 sera than their 293T-grown counterpart, especiallythe TM and TM4 mutants, but still within the Tier 2 spectrum.

As reported previously for NIH45-46 [27], glycan deletion increased thesusceptibility of 426c Env to neutralization by mature CD4bs bNAbs whenthe virus was produced 293T cells (Table 24, FIG. 85A). VRC01 and3BNC117 were ˜10-1000 times more potent against TM and TM4 compared toparental 426c Env. CH103 exhibited moderately improved potency againstDM, TM and TM4. VRC-CH31 exhibited moderately improved potency againstTM4 but was knocked-out by SM and TM, demonstrating a dependency onglycan N276. All 426c Envs produced in 293T cells were resistant toCH235 but were sensitive to CH235.12. (Table 24). Despite 100-fold and1000-fold improved potencies of mature VRC01 against 293T versions of TMand TM4, respectively, no neutralization of these Envs was detected withgermline-reverted VRC01 (Table 24), which agrees with an earlier report[27].

GnTI-production enhanced the susceptibility of parental 426c Env toneutralization by mature VRC01, 3BNC117, VRC-CH31 and CH103 compared towhen the Env was produced in 293T cells, and this susceptibility wasfurther enhanced against one or more glycan-deleted variants of 426cEnv, demonstrating the complementary nature of glycan deletion andGnTI-production for these mature bNAbs (FIG. 85A and Table 24). Incontrast, GnTI-production reduced the susceptibility of parental, DM andTM4 Envs to neutralization by CH235.12 and had little impact on the SMand TM Envs in this case. GnTI-production had little or no impact onmost other mature bNAbs tested (Table 24). A notable exception was a˜100-fold diminished potency of PGT151 (gp120-gp41 epitope), whichagrees with previous findings that PGT151 preferentially bindscomplex-type glycans in microarrays [58] and binds poorly to Env trimerscontaining only high mannose glycans [59]. GnTI-production had nomeasurable impact on VRC34.01, whose epitope overlaps but is distinctfrom that of PGT151 [60].

Neutralization by germline-reverted forms of VRC01-class bNAbs requiresa combination of Man5-enrichment and targeted glycan deletion

Germline-reverted and early intermediates of CD4bs bNAbs were evaluatedfor an ability to neutralize GnTI-version of targeted glycan deleted426c Envs. These tests included near-germline forms of severalVRC01-class bnAbs, which possess a mature HCDR3 region for which thegermline form could not be inferred with existing sequences. They alsoincluded fully reverted germline forms of VRC-CH31, CH103 andCH235/CH235.12. Mature CH235 and CH235.12 are members of the samelineage and exhibit 18% and 90% neutralization breadth, respectively,against a multiclade panel of 199 viruses [2]. Their unmutated commonancestor (UCA) is referred to here as CH235 UCA2.

GnTI-versions of the 426c SM, TM and TM4 Envs were remarkably sensitiveto neutralization by germline-reverted VRC01, with IC50s of 0.99, 2.5and 0.44 μg/ml, respectively (FIG. 85B, Table 24). Germline-revertedVRC01 did not neutralize the 293T versions of these Envs, although apositive deflection was seen against the 293T version of TM4 at thehighest antibody concentrations tested. No neutralization was detectedagainst parental 426c Env produced in either cell type. Thus,germline-reverted VRC01 neutralizes 426c when the Env is bothMan5-enriched and lacking glycan N276. This impact of glycan N276 agreeswith the observation that germline reverted VRC01 binds gp140 trimers of426c.SM and 426c.TM but not 426c.DM (N276 glycan present) produced in293T cells [27]. It has been suggested that germline VRC01 recognizesviruses lacking glycan N276 and that accommodating this glycan leads tobreadth [61]. Our results are consistent with this and suggest that Man5enrichment will further improve germline binding.

Germline forms of other VRC01-class bNAbs also neutralized GnTI-versionsof the TM and TM4 Envs (FIG. 86A). Here, TM was neutralized by germlineforms of VRC07 (IC50=1.6 μg/ml), VRC20 (IC50=4.6 μg/ml) and VRC18(IC50=23 μg/ml). TM4 was neutralized by germline forms of VRC07(IC50=1.7 μg/ml), VRC18 (IC50=17.3 μg/ml), VRC20 (IC50=0.03 μg/ml), and12A12 (IC50=0.63 μg/ml). Thus, 426c.TM and TM4 Envs produced inGnTI-cells are targets for neutralization by some but not allgermline-reverted forms of VRC01-class bNAbs. In order to map thisactivity in sera from vaccine recipients, a known VRC01 resistancemutation, D279K [61], was introduced into 426c.TM. The GnTI-version of426c.TM.D279K Env was highly resistant to germline forms of VRC01, VRC07and VRC20 (FIG. 86B), indicating utility for diagnostic epitope mapping.

Attempts were made to detect neutralization by germline reverted andintermediates of the VRC01-like bNAb, VRC-CH31. Here, 426c.DM was usedbecause the absence of glycan N276 in the single and triple glycanmutant Envs renders 426c resistant to mature VRC-CH31 (FIG. 85A, Table24). No neutralizing activity was detected with the germline-revertedantibody regardless of the Env used; however all four VRC-CH31intermediates neutralized 293T versions of 426c and 426c.DM Envs (FIG.87 ), with the double mutant being slightly more sensitive than parentalEnv. These intermediates exhibited far greater potency (>10-fold) when426c.DM Env was produced in GnTI-cells (FIG. 87 ), suggesting that theGnTI-version of this Env may provide an advantage for engaging anddetecting early intermediates of this lineage.

Neutralization by Germline-Reverted CH103 and Intermediates of CH103 andCH235

The lack of susceptibility of Man5-enriched versions of glycan-deleted426c Envs to neutralization by germline-reverted and intermediates ofCH103 and CH235 (Table 24) led us to test glycan-deleted variants of theautologous transmitted/founder Env (CH0505TF) that evolved and gave riseto CH103 and its CH235-helper lineage [6, 30]. One CH0505TF mutantlacked four glycans at N197, N461/462, N276 and N362 (gly4), whereas theothers lacked three glycans by adding back glycans N197 (gly3.197), N276(gly3.276) or N461 (gly3.461) [62]. CH0505TF naturally lacks glycanN362.

As reported previously [62], 293T versions of parental CH0505TF and allfour glycan mutants were sensitive to mature CH103, CH235 and CH235.12,with the gly4, gly3.276 and gly3.461 Envs often being 10-1000 times moresensitive than the parental and gly3.197 Envs (FIG. 88 , Table 24). The293T versions of gly4, gly3.276 and gly3.461 Envs also were verysensitive to neutralization by intermediates of CH103 and moderatelysensitive to neutralization by the two later intermediates of CH235.GnTI-production generally increased these levels of neutralizationagainst parental CH0505TF and all four glycan mutant Envs (FIG. 88 ,Table 25).

Notably, GnTI-versions of the gly4 and gly3.461 Envs were moderatelysensitive to neutralization by germline-reverted CH103 (IC50=6.4 and10.2 μg/ml, respectively), whereas 293T versions of these Envs were notneutralized (FIG. 88A). GnTI-production also enabled neutralization ofparental CH0505TF Env by the earliest intermediate of CH235 tested(CH23514.v2.4A, IC50=1.4 μg/ml), which was not neutralized when the Envwas produced in 293T cells (FIG. 88B). The glycan mutant Envs, whetherproduced in 293T or GnTI-cells, were highly sensitive to intermediatesof VRC-CH31 (Table 25), and this sensitivity exceeded that seen withglycan mutants of Env 426c (Table 24). Overall these results indicate anadvantage of using engineered CH0505TF rather than 426c Env to detectgermline-reverted CH103 and intermediates of CH103, CH235/CH235.12 andVRC-CH31. Despite this advantage, the modified CH0505TF Envs did notpermit neutralization by germline-reverted VRC01, VRC-CH31 orCH235/CH235.12 (CH235 UCA2) (Table 25).

Neutralization by CH235 UCA2 Requires a Combination of Man5-Enrichmentand Mutation of G458 in gp120

We were interested in developing diagnostic mutants to map theneutralizing activity detected with intermediates of the CH103 and CH235lineages. Two known CD4bs bNAb resistance mutations, N280D (loop D) andG458Y (V5 proximal), were introduced into CH0505TF and assayed as293T-produced Envs against a panel of mature bNAbs (Table 23). N280D andG458Y were strong resistance mutations for VRC01 and 3BNC117. N280D wasa strong resistance mutation for CH235, N6 and VRC-CH31. Neithermutation had a strong impact on CH103, although a 3-fold reduction inneutralization was seen with G458Y. To design a better resistancemutation for CH103, additional point mutations were investigated that incrystal structures are contacts for CH103 but not CD4 (to maintaininfectivity). Three mutations in V5 (N461A, N462A and T463A) had noeffect but a fourth mutation in the CD4-binding loop (S365P) conferredresistance to CH103 (Table 23). The S365P mutation also conferredpartial resistance to VRC-CH31 (Table 23). No mutation reduced theneutralizing activity of CH235.12.

The N280D, G458Y and S365P mutants of CH0505TF were used as GnTI-Envsfor mapping (Table 23 and Table 25). S365P was an effective resistancemutation for CH103 intermediates but was only modestly effective formature CH103. G458Y conferred partial resistance to CH103 intermediates(more complete resistance was seen with the 293T version of this mutantEnv, Table 25). N280D was an effective resistance mutation for the oneintermediate and two mature forms of CH235 that neutralized the parentvirus.

Surprisingly, the G458Y mutation in the context of CH0505TF Env producedin GnTI-cells conferred a high level of susceptibility to neutralizationby CH235 UCA2 and all intermediates of this lineage (FIG. 89A, Table25). This was unexpected for a mutation that confers resistance to otherCD4bs bNAbs [61, 63-67]. CH235 UCA2 did not neutralize the 293T versionof CH0505TF.G458Y Env (Table 25). It also failed to neutralize 293T andGnTI-versions of glycan-deleted CH0505.gly4.G458Y Env (Table 26), whichin the absence of the G458Y mutation was at least 10× more sensitive tomature CH235 and CH235.12 than parental CH0505TF Env (Table 25). Theseobservations demonstrate that germline reverted and intermediates formsof CH235/CH235.12 strongly recognize native functional CH0505TF Env thatcontains a tyrosine (Y) at position 458 and is produced in GnTI-cells.Either Env modification alone was not sufficient. Moreover, deletion ofthree glycans surrounding the CD4bs was detrimental.

Additional amino acid substitutions at position 458 were tested for aneffect similar to tyrosine. CH0505TF Env was sensitive to neutralizationby CH235 UCA2 when position 458 was occupied by phenylalanine (F),tryptophan (W), arginine (R), cysteine (C) and leucine (L), althoughtyrosine remained superior (FIG. 89A, Table 27). Only minor positivedeflections were detected when the position was occupied by lysine (K),serine (S), aspartic acid (D) or glutamic acid (E), the latter two aminoacids being the only ones that are negatively charged (FIG. 89A, Table27). Sensitivity to CH235 UCA2 corresponded to heightened sensitivity tointermediates and mature forms of CH235 (Tables 25 and 27). Nosubstitution substantially altered the neutralization phenotype withHIV-1 sera, although G458D was moderately more sensitive (Table 27).Neutralization of the GnTI-version of CH0505TF.G458Y Env by UCA2 andintermediates of CH235 was negatively impacted by N280D, indicatingdiagnostic utility for mapping (Table 27).

To gain insight into the G458Y impact on CH235 UCA2, the crystalstructure of gp120 in complex with CH235 [2] was examined. As shown inFIG. 89C, glycine G458 in the V5 region of gp120 contacts the aromaticrings of tryptophan (W50) in the CDRH2 of mature CH235. This position inUCAs is isoleucine (I50), which when structurally modeled into thecrystal structure is too small to make interfacial contacts with G458.Based on structural modeling, we hypothesize that replacing the smallglycine residue with tyrosine (Y458), which has a bulky aromatic sidechain, could allow for increased hydrophobic contacts and thus morefavorable binding between the UCA and the G458Y mutant gp120. Indeed,amino acid hydrophobicity at the G458 position was correlated with UCA2neutralization of the mutant viruses (FIG. 89B) suggesting that fillingthe cavity within the gp120-UCA2 interface at position 458 withhydrophobic residues could be a potential structural mechanism forattaining UCA2 neutralization.

We asked whether CH0505TF.G458Y GnTI-Env existed in a more open trimerconformation that is associated with a highly sensitive Tier 1neutralization phenotype [68-70]. The GnTI-version of CH0505TF.G458Y Envwas only 3 times more sensitive to HIV-1 sera than the parental Envgrown in either GnTI- or 293T cells (Table 25). It was also resistant toa panel of antibodies that show preference for Tier 1 Envs(non-neutralizing Abs in Table 25). Moreover, the GnTI-version of ahighly neutralization sensitive Tier 1 variant of CH0505TF Env(CH0505.w4.3) was not neutralized by CH235 UCA2, and was not moresensitive to the intermediates and mature forms of CH235 compared to theGnTI-version of parental CH0505TF Env (Table 25). Overall, thestructural determinants that permit neutralization of Man5-enrichedCH0505TF.G458Y Env by CH235 UCA2 may be less subtle than the open trimerconformation that leads to a Tier 1 neutralization phenotype.

Discussion

Part of the reason why current vaccine immunogens fail to induce bNAbsis that they are unable to stimulate appropriate germline-encoded B cellreceptors. To overcome this limitation, researchers are identifyingnatural and engineered Env proteins that bind germline-reverted forms ofthe bNAbs as partial mimics of the naïve B cell receptors [6, 26, 27,36-38]; such proteins are in early stages of development and it isunclear whether they will initiate correct antibody lineages in humansand wild-type animal models. We sought Env modifications that wouldpermit neutralization by germline forms of CD4bs bNAbs as stringentproof of native envelope engagement by the antibodies. One previousreport described weak neutralization by germline-reverted CH103 againstan early autologous Tier 1 Env [30], which was also observed here(IC50=24 μg/ml, Table 25). Another report described neutralization of426c.SM and TM by VRC01-class bNAb NIH45-46 but only at high antibodyconcentrations (IC50˜100 μg/ml) [27]. We describe Env modifications thatpermit far greater neutralization potency by germline forms of severalCD4bs bNAbs, including VRC01-class (VH1-2) (IC50=0.03 μg/ml),CH235/CH235.12 (VH1-46) (IC50=0.16 μg/ml), and to a lesser extent CH103(VH4-59) (IC50=6.4 μg/ml). This was accomplished by using eithertargeted glycan deletion or mutation of gp120 position 458, combinedwith Man5-enrichment of N-linked glycans that would otherwise be fullyprocessed into complex-type glycans.

Man5-enrichment in GnTI-cells was hypothesized to reduce steric barriersto germline bNAb binding without disrupting native Env conformation.That Man5-enriched Envs were infectious is consistent with previousreports [45, 54] and indicates that native conformation was indeedpreserved. Several mature CD4bs bNAbs were more potent againstMan5-enriched Envs than wild type Envs, while most bNAbs to epitopesoutside the CD4bs were not affected (Tables 24 and 25). One exception isPGT151 (gp120-gp41 epitope), which was negatively impacted byMan5-enrichment. This agrees with previous indications that PGT151requires one or more complex-type glycans [58, 59]. Another exceptionwas the increased potency of V2-apex bNAbs CH01 and PG9 againstglycan-deleted, Man5-enriched variants of CH0505TF (Table 25). Furtherstudies are warranted to determine whether Man5-enrichment might be aviable approach to initiate V2-apex bNAbs.

It was necessary to couple Man5-enrichment with targeted glycan deletionin 426c Env to achieve neutralization by germline-reverted forms ofVRC01-class bNAbs. A simple explanation for why both modifications werenecessary is that not all complex-type glycans acting as steric barriersto germline binding were removed by targeted deletion. Indeed, the lowerglycan density created by targeted sequon removal has potential torelieve steric constraints on α-mannosidases and result in an increasednumber of fully processed complex-type glycans [40-43]. Any additionalcomplex-type glycans generated in this way should remain arrested assmaller Man5 glycoforms when produced in GnTI-cells, thereby affording alower barrier to germline binding.

A remarkable finding was that mutation of G458 in the V5 region of gp120(a CD4 contact residue) enabled germline-reverted and severalintermediates of CH235/CH235.12 to potently neutralize Man5-enrichedCH0505TF Env. Y458 was most effective but other amino acids alsopermitted neutralization. Mutation of this site, usually to negativelycharged aspartic acid (G458D), confers resistance to certain VRC01-classbNAbs [61, 63-67] and was shown here as a G458Y mutation to conferresistance to VRC01 and 3BNC117. Neutralization by germline-reverted andearly intermediates of CH235/CH235.12 required both Man5-enrichment andmutation of G458 without the need for targeted glycan deletion. At themolecular level, G458Y mutation restores a potential contact site in theCDRH2 region of germline-reverted CH235/CH235.12 that is lost when atryptophan (W50) in the mature CDRH2 is reverted to isoleucine in theUCA. Since G458 is highly conserved (>95%) among circulating group M Envsequences [61], and was present in all viral sequences examined from theCH235/CH235.12 donor [30], it seems unlikely that Y458 (or anothersubstitution at this site) contributed to the natural response that gaverise to CH235/CH235.12 in this individual. Indeed, the rarity of non-Gat this position may be part of an evasion mechanism to disfavor theproduction of CH235/CH235.12-like bNAbs. Nonetheless, Man5-enrichedCH0505TF.G458Y Env with all sequons intact may be a potent stimulator ofgermline CH235/CH235.12-like antibodies, and it remains possible thatsuch variants exited in the donor at a low frequency that wentundetected. Similarly, heterogeneity in Env sequon location andoccupation, and in the composition of glycans at occupied sites [49, 51,52] make it possible that other CD4bs bNAb responses in HIV-1 infectedindividuals are driven in part by a subpopulation of Envs that are bothMan5-enriched and lack key sequons.

The Env modifications reported here suggest new avenues to pursue forimmunogen design. For example, immunogens could be tailored to initiatethe CH235/CH235.12 lineage by priming with Man5-enriched CH0505TF.Y458Env protein produced in GnTI-cells and boosting with reverse engineeredimmunogens that contain G458 and a full complement of complex-typeglycans. It will also be of interest to investigate existing VRC01germline-targeting immunogens, such as 426c core [27, 36] and eOD-GT8[38], that are produced in GnTI-cells. Success may depend on combiningthese modifications with other design features, such as closelymimicking native Env structure to assure correct angle of antibodyapproach [71], and circumventing immunologic tolerance [72]. Notably, ithas not been possible to accurately infer the germline version of theCDRH3 region of VRC01-class bNAbs with existing sequences. Thus, whiledetection of neutralizing activity by the germline form of VRC01 usedhere is encouraging, additional Env modifications might be needed toadequately engage true VRC01-class germline B cells.

The modified Envs described here have additional value by enablingdetection of early precursors of CD4bs bNAbs induced by candidateimmunogens. Detection would be based on functional neutralizing activityin a high throughput assay and would complement other technologies, suchas antigen-specific memory B cell sorting and immunoglobulin sequenceanalyses. Until the technology is refined to capture a wider range ofCD4bs bNAb precursors, negative neutralizing activity would notnecessarily mean that precursors are absent. Additional efforts areneeded to be more inclusive of the full range of CD4bs bNAbs and toenable detection of early precursors of bNAbs to other epitopes inneutralization assays. The insights provided here should facilitatethese efforts as they relate to both immune monitoring and immunogendesign.

Methods

Cells

TZM-bl, 293T/17 and 293S/GnTI-cells were maintained in Dulbecco'sModified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS)and gentamicin (50 μg/ml) in vented T-75 culture flasks(Corning-Costar). Cultures were incubated at 37° C. in a humidified 5%CO2-95% air environment. Cell monolayers were split 1:10 at confluenceby treatment with 0.25% trypsin, 1 mM EDTA.

Antibodies and HIV-1 Sera

The monoclonal antibodies used in this study have been previouslydescribed: CD4bs bNAbs VRC01, VRC03, VRC04, VRC07, VRC-18b, VRC20,VRC23, 12A12 [8-10, 24], 3BNC117, 3BNC60 [7], VRC-CH31 [73], N6 [5],HJ16 [3] and IgG1b12 [74]; high mannose glycan-specific bNAb 2G12 [75];gp41 membrane proximal external region (MPER)-specific bNAbs 2F5, 4E10[76], 10E8 [77] and DH511.2_K3 [78]; V2-apex bNAbs PG9, PG16 [79], CH01[73] and PGDM1400 [80]; V3-glycan bNAbs PGT121, PGT128 and 10-1074 [56,81]; gp41-gp120 interface bNAbs PGT151 [58] and VRC34.01 [60]. VRC01,VRC34.01 and 10E8 were produced by the Vaccine Research Center, NIH. N6was obtained from Dr. Mark Connors. 3BNC117, 3BNC60 and 10-1074 wereobtained from Dr. Michel Nussenzweig. VRC-CH31 and CH01 were produced byCatalent Biologics (Madison, Wis.). DH511.2_K3 was produced by the HumanVaccine Institute, Duke University Medical Center. HJ16 was obtainedfrom Dr. Davide Corti. IgG1b12, 2G12, 2F5, 4E10, PG9 and PG16 werepurchased from Polymun Scientific (Klosterneuburg, Austria). PGDM1400,PGT121, PGT128 and PGT151 were a kind gift from Dr. Dennis Burton.

In addition to these mature bNAbs we utilized UCAs, intermediates andmature forms of CH103, CH235/CH235.12 [2, 30] and VRC-CH31 [10], whichwere produced by the Human Vaccine Institute, Duke University MedicalCenter, Durham, N.C. The unmutated common ancestor (UCA) sequence forthe CH235/CH235.12 lineage used in this study differs by one amino acidfrom the UCA described previously [2]. The UCA used here, which we referto as CH235 UCA2, has a methionine in the 4th position of the lightchain in place of a leucine in the previously described UCA version.Other antibodies included germline-reverted forms of the VRC01-classbNAbs VRC01, VRC03, VRC04, VRC07, VRC18b, VRC20, VRC23, 12A12 and3BNC117 [9, 10, 24, 27], which were produced at the Vaccine ResearchCenter, NIH. These latter germline-reverted antibodies possess a matureHCDR3 region, which could not be inferred with existing sequences.

Neutralization Tier phenotyping was performed with serum pools fromindividuals in southern Africa (South Africa, Malawi and Tanzania) whoparticipated in a CHAVI study of chronic HIV-1 infection (CHAVI samples0406, 0060, 0642, 0293, 0598, 0537, 0468, 0461, 0382 and 0134). Thesestudy subjects had all been infected for at least three years. Samplesfrom 6-10 time points collected over 8-60 months were pooled on aper-subject basis and heat-inactivated for 30 minutes at 56° C. Fordeeper interrogation of neutralization phenotype, a set of monoclonalantibodies was used that show a strong preference for Tier 1 viruses.This set included V3-specific antibodies 2219, 2557, 3074, 3869, 447-52Dand 838-D, and the CD4bs antibodies 654-30D, 1008-30D, 1570D, 729-30Dand F105, all produced by Drs. Susan Zolla-Pazner and Miroslaw K. Gornyat New York University and the Veterans Affairs Medical Center, NewYork, N.Y.

Pseudotyping Envs

Full-length functional HIV-1 Envs were used for virus pseudotyping.Previous reports described Envs for strains CE1176 [82], WITO [83],TRO.11 [83], CH0505TF and CH0505.w4.3 [2]. Glycan deleted EnvsCH0505TF.gly4, CH0505TF.gly197, CH0505TF.gly3.276 and CH0505TF.gly3.461were described by Zhou et al. [62]. Envs for 426c and the glycan deletedvariants 426c.SM, 426c.DM and 426c.TM were described by McGuire et al.[27]. In some cases N280D, G458Y and S365P mutations were introduced bysite-directed mutagenesis as described [84].

Transfection

Env-pseudotyped viruses were produced in either 293T/17 or 293SGnTI-cells (American Type Culture Collection) as described [85]. 293SGnTI-cells lack the enzyme N-acetylglucosaminyltransferase and have beenshown to yield HIV-1 Envs that contain Man6-9 glycoforms and areenriched for under-processed Man5 glycoforms in place of complex glycans[45, 54]. Env-pseudoviruses were generated by transfecting exponentiallydividing 293T/17 or 293 S/GnTI-cells (5×106 cells in 12 ml growth mediumin a T-75 culture flask) with 4 μg of rev/env expression plasmid and 8μg of an env-deficient HIV-1 backbone vector (pSG3ΔEnv), using Fugene 6transfection reagent. Cells were washed after 3-8 hours and incubated infresh growth medium without transfection reagents.Pseudovirus-containing culture supernatants were harvested 2 days aftertransfection, filtered (0.45 μm), and stored at −80° C. in 1 mlaliquots. Infectivity was quantified in TZM-bl cells by performingserial fivefold dilutions of pseudovirus in quadruplicate wells in96-well culture plates in a total volume of 100 μl of growth medium fora total of 11 dilution steps. Freshly trypsinized cells (10,000 cells in100 μl of growth medium containing 75 μg/ml DEAE-dextran) were added toeach well, and the plates were incubated at 37° C. in a humidified 5%CO2-95% air environment. After a 48-hour incubation, 100 μl of culturemedium was removed from each well and 100 μl of Britelite reagent wasadded to the cells. After a 2-min incubation at room temperature toallow cell lysis, 150 μl of cell lysate was transferred to 96-well blacksolid plates (Corning-Costar) for measurements of luminescence using aVictor 3 luminometer (Perkin-Elmer Life Sciences, Shelton, Conn.). Adilution of virus that results in 50,000-250,000 relative luminescenceunits (RLUs) was used for neutralization assays.

Neutralization Assay

Neutralization assays were performed in TZM-bl cells (NIH AIDS Researchand Reference Reagent Program) as described [85]. Briefly, apre-titrated dose of Env-pseudotyped virus was incubated with serial3-fold dilutions of test sample in duplicate in a total volume of 150 μlfor 1 hr at 37° C. in 96-well flat-bottom culture plates. Freshlytrypsinized cells (10,000 cells in 100 μl of growth medium containing 20μg/ml DEAE dextran) were added to each well. One set of control wellsreceived cells+virus (virus control) and another set received cells only(background control). After 48 hours of incubation, the cells were lysedby the addition of Britelite (PerkinElmer Life Sciences) and threequarters of the cell lysate was transferred to a 96-well black solidplate (Costar) for measurement of luminescence. Neutralization titersare either the serum dilution (ID50) or antibody concentration (IC50) atwhich relative luminescence units (RLU) were reduced by 50% compared tovirus control wells after subtraction of background RLUs.

Structural Modeling and Analysis

Structural modeling of mutations in the CH235 gp120 complex (PDB: 5F9W)[2] was performed with the PyMOL Molecular Graphics System, Version 1.8Schrödinger, LLC (http://www.pymol.org) using the mutagenesis wizard andplacing mutated residues in the rotamer state corresponding to theminimum strain value. Hydrophobicity scores were assigned to amino acidsusing the Wimley-White whole-residue octanol scale [86]. For position458 mutants that had neutralization curves that did not reach 50%neutralization at the highest concentration (10 mg/ml), the IC50 valuewas set to 25 for the regression analysis.

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Example 11

TABLE 23 Epitope mapping identifies a G458Y mutation in the context ofGnTI-CH0505TF Env as a potential germline-targeting feature for theCH235 lineage. Envs Produced in 293T Cells:¹ IC50 (μg/ml) in TZM-blANTIBODY CH0505TF CH0505TF.N280D CH0505TF.G458Y CH0505TF.S365P CH103 1.91.2 5.8 >50 CH235 0.3 >25 0.1 0.6 CH235.12 0.04 0.06 0.01 0.03 VRC010.1 >25 >25 0.1 3BNC117 0.03 >25 >25 0.01 N6 0.06 >17 0.02 0.02 VRC-CH310.03 >25 <0.01 1.2 Envs Produced in 293S GnTI Cells:² IC50 (μg/ml) inTZM-bl ANTIBODY CH0505TF CH0505TF.N280D CH0505TF.G458Y CH0505TF.S365PCH103_UCA1.1_4A >50 >50 >50 >50 CH103_UCAGrand5 >50 >50 >50 >50CH103_IA_9_4A >50 >50 >50 >50 CH103_IA_8_4A 0.55 0.85 2.0 >50CH103_IA_7_4A 0.13 0.4 0.9 >50 CH103_IA_6_4A 0.64 2.8 8.7 >50CH103_IA_5_4A 0.46 0.18 12.7 >30 CH103_IA_4_4A 0.19 0.22 1.0 >50 CH1030.57 0.24 0.44 1.7 CH235 UCA2 >25 >25 0.16 >25 CH235_I4_v2_4A/293i1.4 >50 0.08 26 CH235_I3_v2_4A 0.12 >50 0.03 0.16 CH235VH_I1_v2_4A/293i0.05 >50 <0.02 0.04 CH235 0.03 3.4 <0.02 0.02 CH235.12 0.02 0.02 <0.020.01 ¹Highlight values in the top section of the table are the mostdramatic cases of resistance-mediating effects. ²Highlighted values inthe bottom section of the table are the remarkable neutralizationpotencies seen with UCAs and intermediates of CH235.

Example 11

TABLE 24 Neutralization properties of parental and targeted glycandeleted variants of Env 426c produced in either 293T or 293S GnTI⁻cells. Tier Phenotyping: ID50 (dilution) in TZM-bl 293T VIRUSES ReagentEpitope 426c 426c.SM 426c.DM 426c.TM 426c.TM4 HIV-1 sera: CHAVI-0406Poly- 20 20 20 86 58 clonal CHAVI-0060 Poly- 40 31 31 52 49 clonalCHAVI-0642 Poly- 55 68 51 129 67 clonal CHAVI-0293 Poly- 20 20 20 113 87clonal CHAVI-0598 Poly- 199 264 225 451 229 clonal Geometric mean titer45 47 43 124 82 293S GnTI− VIRUSES Reagent Epitope 426c 426c.SM 426c.DM426c.TM 426c.TM4 HIV-1 sera: CHAVI-0406 Poly- 172 201 230 338 351 clonalCHAVI-0060 Poly- 100 144 95 338 135 clonal CHAVI-0642 Poly- 282 339 169519 309 clonal CHAVI-0293 Poly- 26 46 42 543 261 clonal CHAVI-0598 Poly-963 1544 1152 3011 1124 clonal Geometric mean titer 165 234 178 627 336Monoclonal Antibodies: IC50 (μg/ml) in TZM-bl 293T VIRUSES ReagentEpitope 426c 426c.SM 426c.DM 426c.TM 426c.TM4 Non-neutralizing Abs: 2219V3 >25 >25 >25 >25 >25 2557 V3 >25 >25 >25 >25 >25 3074V3 >25 >25 >25 >25 >25 3869 V3 >25 >25 >25 >25 >25 447-52DV3 >25 >25 >25 >25 >25 838-12D V3 >25 >25 >25 >25 >25 654-30DCD4bs >25 >25 >25 >25 >25 1008-30D CD4bs >25 >25 >25 >25 >25 1570DCD4bs >25 >25 >25 >25 >25 729-30D CD4bs >25 >25 >25 >25 >25 F105CD4bs >25 >25 >25 >25 >25 Mature bNAbs: 2G12 glycan >25 >25 >25 >25 >252F5 MPER >25 >25 >25 >25 >25 4E10 MPER 3.32 1.52 1.00 0.98 1.66 10E8MPER 0.8 1.26 1.11 1.52 0.53 DH511.2_K3 MPER 0.8 0.77 1.50 1.06 0.56CH01 V2-gly >25 >25 >25 >25 >25 PG9 V2-gly >5 >5 >5 >5 >5 PG16V2-gly >5 >5 >5 >5 >5 PGDM1400 V2-gly >25 >25 >25 >25 >5 PGT121V3-gly >5 >5 >5 >5 >5 PGT128 V3-gly >5 >5 >5 >5 >5 10-1074 V3-gly 0.050.12 0.10 0.16 0.06 PGT151 gp120/ 0.01 0.01 0.01 0.01 0.01 gp41 VRC34.01gp120/ 0.08 0.06 0.09 0.08 0.13 gp41 b12 CD4bs >25 >25 >25 >25 >25 HJ16CD4bs >25 >25 >25 >25 >25 3BNC117 CD4bs 0.20 0.24 0.13 0.01 0.003VRC-CH31 CD4bs 0.62 >25 0.73 >25 0.15 CH103 CD4bs >40 >40 6.1 5.2 3.7CH235 CD4bs >50 >50 >50 >50 >25 CH235.12 CD4bs 8.95 1.08 0.66 0.09 0.1VRC01 CD4bs 2.20 0.39 0.41 0.03 0.002 VRC03 CD4bs nt nt nt nt 0.003VRC04 CD4bs nt nt nt nt 0.013 VRC07 CD4bs nt nt nt nt <0.002 VRC018CD4bs nt nt nt nt 0.005 VRC20 CD4bs nt nt nt nt <0.002 (VRC-PG20) VRC23CD4bs nt nt nt nt 0.082 12A12 CD4bs nt nt nt nt 0.003 UCA andintermediate Abs: VRC01gHvgLv CD4bs >50 >50 >50 >50 >25 VRC03gHvgLvCD4bs nt nt nt nt >25 VRC04gHvgLv CD4bs nt nt nt nt >25 VRC07gHvgLvCD4bs nt nt nt nt >25 VRC18gHvgLv CD4bs nt nt nt nt >25 VRC20 (VRC-CD4bs nt nt nt nt 2.39 PG20)gHvgLv VRC23gHvgLv CD4bs nt nt nt nt >2512A12gl CD4bs nt nt nt nt >25 3BNC117gl CD4bs nt nt nt nt >25CH103_UCA1.1_4A CD4bs >50 nt nt >50 >25 CH103_UCAGrand5 CD4bs >50 ntnt >50 >25 CH103_IA_9_4A CD4bs >50 nt nt >50 >25 CH103_IA_8_4A CD4bs >50nt nt >50 >25 CH103_IA_7_4A CD4bs >50 nt nt >50 >25 CH103_IA_6_4ACD4bs >50 nt nt >50 >25 CH103_IA_5_4A CD4bs >20 nt nt >20 >25CH103_IA_4_4A CD4bs >50 nt nt >50 >25 CH235 UCA2 CD4bs >50 nt nt >50 >25CH235_I4_v2_4A CD4bs >50 nt nt >50 >25 CH235_I3_v2_4A CD4bs >50 ntnt >50 >25 CH235VH_I1_v2_4A CD4bs >50 nt nt >50 >25 VRC-CH31 CD4bs >50nt >50 nt >25 AbCH3X_UCA VRC-CH31 CD4bs 1.3 nt 0.6 nt 0.02 AbCH3X_I4VRC-CH31 CD4bs 1.4 nt 0.8 nt 0.01 AbCH3X_I3 VRC-CH31 CD4bs 1.5 nt 0.9 nt0.07 AbCH3X_I2 VRC-CH31 CD4bs 1.9 nt 1.15 nt 0.15 AbCH3X_I1 293S GnTI−VIRUSES Reagent Epitope 426c 426c.SM 426c.DM 426c.TM 426c.TM4Non-neutralizing Abs: 2219 V3 >25 >25 >25 >25 >25 2557V3 >25 >25 >25 >25 >25 3074 V3 >25 23 >25 >25 >25 3869V3 >25 >25 >25 >25 >25 447-52D V3 >25 >25 >25 >25 >25 838-12DV3 >25 >25 >25 >25 >25 654-30D CD4bs >25 >25 >25 >25 >25 1008-30DCD4bs >25 >25 >25 >25 >25 1570D CD4bs >25 >25 >25 >25 >25 729-30DCD4bs >25 >25 >25 >25 >25 F105 CD4bs >25 >25 >25 >25 >25 Mature bNAbs:2G12 glycan >25 >25 >25 >25 >25 2F5 MPER >25 >25 >25 >25 >25 4E10 MPER 43.8 3.7 4 1.6 10E8 MPER 0.35 0.28 0.45 0.51 0.19 DH511.2_K3 MPER 0.850.87 0.75 1.2 0.56 CH01 V2-gly >25 >25 >25 >25 >25 PG9V2-gly >5 >5 >5 >5 >5 PG16 V2-gly >5 >5 >5 >5 >5 PGDM1400V2-gly >5 >5 >5 >5 >5 PGT121 V3-gly 2.5 4.2 3.4 3.4 >5 PGT128 V3-gly4.3 >5 >5 >5 >5 10-1074 V3-gly 0.03 0.04 0.03 0.03 0.02 PGT151 gp120/1.6 1.9 2.2 2.5 2.9 gp41 VRC34.01 gp120/ 0.05 0.07 0.04 0.07 0.07 gp41b12 CD4bs >25 >25 >25 >25 >25 HJ16 CD4bs >25 >25 >25 >25 >25 3BNC117CD4bs 0.02 0.01 0.006 0.003 <0.001 VRC-CH31 CD4bs 0.04 0.7 0.02 1.6 0.01CH103 CD4bs 5.3 2.2 0.63 0.09 0.48 CH235 CD4bs >25 >25 >25 >25 >25CH235.12 CD4bs >25 0.26 >25 0.07 24.2 VRC01 CD4bs 0.19 0.04 0.04 0.0150.007 VRC03 CD4bs 0.014 0.003 0.005 0.003 <0.002 VRC04 CD4bs 0.18 0.020.05 0.01 <0.002 VRC07 CD4bs 0.07 0.02 0.05 0.009 <0.002 VRC018 CD4bs0.06 0.01 0.02 0.006 <0.002 VRC20 CD4bs 0.015 0.004 0.007 0.005 0.008(VRC-PG20) VRC23 CD4bs 0.19 0.1 0.06 3.1 <0.002 12A12 CD4bs 0.06 0.010.01 0.01 <0.002 UCA and intermediate Abs: VRC01gHvgLv CD4bs >500.99 >50 2.5 0.44 VRC03gHvgLv CD4bs >25 >25 >25 >25 >25 VRC04gHvgLvCD4bs >25 >25 >25 >25 >25 VRC07gHvgLv CD4bs >25 0.76 >25 1.6 1.7VRC18gHvgLv CD4bs >25 >22 >25 23 17.3 VRC20 (VRC- CD4bs >25 10.3 >25 4.60.03 PG20)gHvgLv VRC23gHvgLv CD4bs >25 >25 >25 >25 >25 12A12glCD4bs >25 >25 >25 >25 0.63 3BNC117gl CD4bs >25 >25 >25 >25 >25CH103_UCA1.1_4A CD4bs nt nt nt >50 >25 CH103_UCAGrand5 CD4bs nt ntnt >50 >25 CH103_IA_9_4A CD4bs nt nt nt >50 >25 CH103_IA_8_4A CD4bs ntnt nt >50 >25 CH103_IA_7_4A CD4bs nt nt nt >50 >25 CH103_IA_6_4A CD4bsnt nt nt >50 >25 CH103_IA_5_4A CD4bs nt nt nt >20 >20 CH103_IA_4_4ACD4bs nt nt nt >50 >25 CH235 UCA2 CD4bs nt nt nt >50 >25 CH235_I4_v2_4ACD4bs nt nt nt >50 >25 CH235_I3_v2_4A CD4bs nt nt nt >50 >25CH235VH_I1_v2_4A CD4bs nt nt nt >50 >25 VRC-CH31 AbCH3X_UCA CD4bs ntnt >50 nt >25 VRC-CH31 CD4bs nt nt 0.018 nt <0.011 AbCH3X_I4 VRC-CH31CD4bs nt nt 0.021 nt <0.011 AbCH3X_I3 VRC-CH31 CD4bs nt nt 0.02 nt<0.011 AbCH3X_I2 VRC-CH31 CD4bs nt nt 0.032 nt <0.011 AbCH3X_I1 nt, nottested 426c.SM (N276D) 426c.DM (N460D. N463D) 426c.TM (N276D. N460D.N463D) 426cTM4 (S278R. G471S. N460D. N463D)

Example 11

TABLE 25 Neutralization properties of parental and targeted glycanvariants of CH0505TF produced in 293T and 293S GnTI⁻ cells. TierPhenotyping: ID50 (dilution) in TZM-bl 293T VIRUSES CH0505TF. CH0505TF.CH0505TF. CH0505TF. CH0505TF. CH0505. Reagent Epitope CH0505TF gly4gly3.197 gly3.276 gly3.461 G458Y w4.3 HIV-1 sera: CHAVI-0537 Poly- 56352 266 456 211 711 23012 clonal CHAVI-0468 Poly- 980 55704 13369 2448518158 3513 18810 clonal CHAVI-0461 Poly- 15 614 154 503 70 462 16739clonal CHAVI-0383 Poly- 68 990 595 262 459 675 13744 clonal CHAVI-0134Poly- 2233 7505 2106 9589 5340 43740 9533 clonal GMT 166 2456 927 1698920 2025 15685 293S GnTI− VIRUSES CH0505TF. CH0505TF. CH0505TF.CH0505TF. CH0505TF. Reagent Epitope CH0505TF gly4 gly3.197 gly3.276gly3.461 G458Y HIV-1 sera: CHAVI-0537 Poly- 148 990 794 897 843 307clonal CHAVI-0468 Poly- 3245 66767 25872 19190 42286 43740 clonalCHAVI-0461 Poly- 71 1796 742 1792 238 98 clonal CHAVI-0383 Poly- 1191228 723 709 685 183 clonal CHAVI-0134 Poly- 38 600 15 514 338 113clonal GMT 173 2446 698 1622 1145 486 Monoclonal Antibodies: IC50(μg/ml) in TZM-bl 293T VIRUSES CH0505TF. CH0505TF. CH0505TF. CH0505TF.CH0505TF. CH0505. Epitope CH0505TF gly4 gly3.197 gly3.276 gly3.461 G458Yw4.3 Non-neutralizing Abs: 2219 V3 >25 >25 >25 >25 >25 >25 >25 2557V3 >25 >25 >25 >25 >25 >25 >25 3074 V3 >25 7.2 >25 14.1 7.3 9.4 0.023869 V3 >25 >25 >25 >25 >25 >25 3.1 447-52D V3 >25 >25 >25 >25 >25 >253.7 838-12D V3 >25 >25 >25 >25 >25 >25 14.9 654-30DCD4bs >25 >25 >25 >25 >25 >25 0.2 1008-30D CD4bs >25 >25 >25 >25 >25 >250.7 1570D CD4bs >25 >25 >25 >25 >25 >25 0.15 729-30DCD4bs >25 >25 >25 >25 >25 >25 F105 CD4bs >25 >25 >25 >25 >25 >25 10.8bNAbs: 2G12 glycan >25 >25 >25 >25 >25 >25 >50 2F5MPER >25 >25 >25 >25 >25 >25 >25 4E1O MPER 22 14 21 17 16 8 >50 1OE8MPER 4.4 2.7 3.4 3.3 3.3 1.1 1.1 DH511.2_K3 MPER 3.4 2.1 3.1 2.9 2.8 0.60.02 CH01 V2-gly 4.1 8.8 3.8 5.7 7.7 1 >20 PG9 V2-gly 0.19 0.21 0.240.17 0.22 0.1 1.8 PG16 V2-gly 0.09 0.1 0.05 0.1 0.1 0.03 0.03 PGDM1400V2-gly 0.006 0.016 0.01 0.02 0.04 0.006 PGT121V3-gly >5 >5 >5 >5 >5 >5 >20 PGT128 V3-gly >5 >5 >5 >5 >5 >5 >10 10-1074V3-gly >25 >25 >25 >25 >25 >25 >25 PGT151 gp120/ 0.01 0.01 0.01 0.0070.01 0.01 >25 gp41 VRC34.01 gp120/ 0.55 0.94 2.1 0.62 0.84 0.21 >25 gp41HJ16 CD4bs >25 >25 >25 >25 >25 >25 >25 b12 CD4bs 0.98 0.004 0.68 0.0070.011 0.1 <0.02 VRC01 CD4bs 0.1 0.002 0.023 0.004 1.98 >25 0.05 3BNC117CD4bs 0.03 0.001 0.005 0.001 0.002 >25 0.02 VRC-CH31 CD4bs 0.03 0.0020.01 0.002 0.09 0.023 0.05 CH103 CD4bs 1.9 0.002 1.6 0.014 0.014 6.40.31 CH235 CD4bs 0.3 0.022 0.16 0.034 0.086 0.1 0.4 CH235.12 CD4bs 0.040.001 0.008 0.001 0.003 0.014 0.04 N6 CD4bs 0.06 0.001 0.01 0.003 0.0040.02 0.01 Germline and intermediate Abs: VRC01/gHvgLvCD4bs >50 >50 >50 >50 >50 >50 >50 CH103_UCA1.1_4ACD4bs >50 >50 >50 >50 >50 >50 24.1 CH103_UCAGrand5CD4bs >50 >50 >50 >50 >50 >50 >50 CH103_IA_9_4ACD4bs >50 >50 >50 >50 >50 >50 >50 CH103_IA_8_4A CD4bs 25.2 0.002 8.50.002 0.013 >50 0.95 CH103_IA_7_4A CD4bs 4.7 0.002 5.9 0.001 0.012 >500.2 CH103_IA_6_4A CD4bs >50 0.032 >50 0.047 0.008 >50 1.5 CH103_IA_5_4ACD4bs 7.6 0.003 2.8 0.002 0.018 >50 0.19 CH103_IA_4_4A CD4bs 3.2 0.0010.97 0.004 0.008 >50 0.25 CH235 UCA2 CD4bs >50 >50 >50 >50 >50 >25 >25CH235_I4_v2_4A CD4bs >50 >50 >50 >50 >50 >25 >25 CH235_I3_v2_4A CD4bs5.3 4.1 >50 1.5 8.6 1.2 3.0 CH235VH_I1_v2_4A CD4bs 0.85 0.23 1.8 0.281.6 0.27 0.6 VRC-CH31 AbCH3X_UCA CD4bs >25 >25 >25 >25 >25 >25 >25VRC-CH31 AbCH3X_I4 CD4bs 0.052 <0.002 0.015 0.002 1.096 0.361 ntVRC-CH31 AbCH3X_I3 CD4bs 0.063 <0.002 0.02 0.002 2.031 0.143 nt VRC-CH31AbCH3X_I2 CD4bs 0.053 <0.002 0.022 0.002 0.297 0.067 nt VRC-CH31AbCH3X_I1 CD4bs 0.088 <0.002 0.031 0.003 >5 1.35 nt 293S GnTI− VIRUSESCH0505TF. CH0505TF. CH0505TF. CH0505TF. CH0505TF. CH0505. EpitopeCH0505TF gly4 gly3.197 gly3.276 gly3.461 G458Y w4.3 Non-neutralizingAbs: 2219 V3 >25 >25 >25 >25 >25 >25 2557 V3 >25 >25 >25 >25 >25 >253074 V3 >25 5.6 >25 4.8 10.2 >25 3869 V3 >25 >25 >25 >25 >25 >25 447-52DV3 >25 >25 >25 >25 >25 >25 838-12D V3 >25 >25 >25 >25 >25 >25 654-30DCD4bs >25 >25 >25 >25 >25 >25 1008-30D CD4bs >25 >25 >25 >25 >25 >251570D CD4bs >25 >25 >25 >25 >25 >25 729-30DCD4bs >25 >25 >25 >25 >25 >25 F105 CD4bs >25 >25 >25 >25 >25 >25 bNAbs:2G12 glycan >25 >25 >25 >25 >25 >25 2F5 MPER >25 >25 >25 >25 >25 >254E1O MPER 10 12.3 14 13 12 10 1OE8 MPER 2.2 2.2 4 2.3 2.5 2.1 DH511.2_K3MPER 0.95 1.1 2.5 1.7 1.6 1.2 CH01 V2-gly 0.8 0.1 0.22 1 0.09 0.1 PG9V2-gly 0.11 0.02 0.06 0.04 0.05 0.04 PG16 V2-gly 0.08 0.02 0.03 0.020.02 0.01 PGDM1400 V2-gly 0.007 0.007 0.003 0.004 0.008 0.002 PGT121V3-gly >5 >5 >5 >5 >5 >5 PGT128 V3-gly >5 >5 >5 >5 >5 >5 10-1074V3-gly >25 >25 >25 >25 >25 >25 PGT151 gp120/ >5 0.13 >5 >5 >5 >5 gp41VRC34.01 gp120/ 0.1 0.14 0.17 0.14 0.17 0.03 gp41 HJ16CD4bs >25 >25 >25 >25 >25 >25 b12 CD4bs 0.61 0.001 0.26 0.01 0.02 0.22VRC01 CD4bs 0.03 0.001 0.009 0.003 0.015 0.91 3BNC117 CD4bs 0.01 0.0010.004 0.001 0.002 18 VRC-CH31 CD4bs 0.04 0.001 0.01 0.001 0.007 0.06CH103 CD4bs 0.57 0.005 0.19 0.007 0.009 0.44 0.18 CH235 CD4bs 0.03 0.0040.006 0.02 0.011 <0.02 0.03 CH235.12 CD4bs 0.015 0.001 0.003 0.005 0.0020.003 0.01 N6 CD4bs 0.016 0.001 0.003 0.004 0.002 0.008 Germline andintermediate Abs: VRC01/gHvgLv CD4bs >50 >50 >50 >50 >50 >50 >50CH103_UCA1.1_4A CD4bs >50 6.4 >50 >50 10.2 >50 14.5 CH103_UCAGrand5CD4bs >50 >50 >50 >50 >50 >50 >50 CH103_IA_9_4ACD4bs >50 >50 >50 >50 >50 >50 >50 CH103_IA_8_4A CD4bs 0.55 0.001 0.140.002 0.003 2 0.21 CH103_IA_7_4A CD4bs 0.13 0.001 0.02 0.002 0.003 0.90.08 CH103_IA_6_4A CD4bs 0.64 0.001 2.2 0.001 0.002 8.7 1.04CH103_IA_5_4A CD4bs 0.46 0.002 0.48 0.003 0.005 12.7 0.09 CH103_IA_4_4ACD4bs 0.19 0.001 0.035 0.001 0.002 1 0.08 CH235 UCA2CD4bs >50 >50 >50 >50 >50 0.16 >25 CH235_I4_v2_4A CD4bs1.4 >50 >50 >50 >50 0.08 1.7 CH235_I3_v2_4A CD4bs 0.12 0.8 0.018 0.350.44 0.03 0.1 CH235VH_I1_v2_4A CD4bs 0.05 0.24 0.022 0.1 0.31 <0.02 0.04VRC-CH31 CD4bs >25 >25 >25 >25 >25 >25 nt AbCH3X_UCA VRC-CH31 CD4bs0.008 <0.002 0.004 0.002 0.025 0.004 nt AbCH3X_I4 VRC-CH31 CD4bs 0.01<0.002 0.003 0.002 0.06 0.004 nt AbCH3X_I3 VRC-CH31 CD4bs 0.009 <0.0020.004 0.002 0.01 0.002 nt AbCH3X_I2 VRC-CH31 CD4bs 0.014 <0.002 0.0050.003 0.357 0.009 nt AbCH3X_I1

Example 11

TABLE 26 CH235 UCA2 does not neutralize targeted glycan deletedCH0505TF.G458Y. IC50 (μg/ml) in TZM-bl cells 293T 293S GnTI⁻CH0505TF.gly4. CH0505TF.gly4. Reagent Epitope G458Y G458Y CH235 UCA2CD4bs >10 >10 CH235_I4_v2_4A CD4bs >10 >10 CH235_I3_v2_4A CD4bs 0.370.07 CH235VH_I1_v2_4A CD4bs 0.016 0.005 CH235 CD4bs 0.064 <0.005CH235.12 CD4bs <0.005 <0.005 Positive values are shown in boldface type

Example 11

TABLE 27 G458 mutations that permit neutralization by CH235 UCA2. IC50(μg/ml) in TZM-bl cells Mutation in CH0505TF (Env-pseudotyped virusesproduced 293S GnTI⁻ cells) Antibody Epitope G458Y G458F G458W G458RG458K CH235 UCA2 CD4bs 0.200 0.746 1.069 0.916 >10 CH235_I4_v2_4A CD4bs0.146 0.249 0.012 0.238 0.798 CH235_I3_v2_4A CD4bs 0.017 0.011 0.0050.025 0.025 CH235VH_I1_v2_4A CD4bs 0.009 0.009 0.006 0.012 0.019 CH235CD4bs <0.005 <0.005 <0.005 <0.005 0.022 HIV-1 Serum CHAVI-0537Polyclonal 124 157 174 160 192 CHAVI-0468 Polyclonal 8257 9993 161535371 4533 CHAVI-0461 Polyclonal 72 109 108 149 160 CHAVI-0383 Polyclonal155 216 236 224 209 CHAVI-0134 Polyclonal 80 121 85 83 102 GMT¹ 247 339361 299 312 Antibody Epitope G458C G458L G458S G458D G458E G458Y.N280DCH235 UCA2 CD4bs 0.391 0.555 >10 >10 >10 >10 CH235_I4_v2_4A CD4bs 0.1030.169 0.901 6.542 >10 >10 CH235_I3_v2_4A CD4bs 0.026 0.025 0.025 0.0660.036 0.771 CH235VH_I1_v2_4A CD4bs 0.017 0.018 0.013 0.033 0.014 0.260CH235 CD4bs 0.009 0.015 0.005 0.011 0.012 2.691 HIV-1 Serum CHAVI-0537Polyclonal 186 293 219 346 197 nt CHAVI-0468 Polyclonal 8854 7048 10905141222 14778 nt CHAVI-0461 Polyclonal 149 274 124 184 124 nt CHAVI-0383Polyclonal 196 154 255 297 301 nt CHAVI-0134 Polyclonal 129 81 112 21678 nt GMT¹ 362 371 385 896 385 nt ¹GMT, geometric mean titer ofpolyclonal sera

Example 12: CH235UCA Versions and Purification

Several CH235UCAs were deduced, made and used in experiments throughoutthis application.

Table 28 shows a summary of the different CH235UCAs. Sequences arereferenced in Examples 8 and 11, and shown in FIG. 59 .

CH235 UCA Name VH VL Lot CH235UCA_LL CH235HUCA_4A CH235KUCA 217SJA DH235UCAtk_v2_4A/293i DH235VH_UCAtk_v2_4A DH235VK_UCAtk 5RKK (also referredto as CH235UCAtk_v2 referenced in Ex 8) DH235UCAtkLL_v3_4A/293iDH235VH_UCAtk_v2_4A CH235KUCA 48EML (also referred to as CH235UCAtkLL_v3and CH235UCA2)

In some experiments, e.g. experiments depicted in Examples 10 and 11CH235 UCA forms were recombinantly expressed and purified without sizeexclusion chromatography step.

FIG. 41 shows that in the absence of size exclusion chromatography step,high molecular weight forms of CH235UCAtkLL_v3 are observed in additionto the main antibody peak. The main peak was isolated and FIG. 42 showsa single antibody peak after size exclusion chromatography purification.

Some experiments in Example 13 compared the properties of theSEC-purified antibody and non-SEC purified CH235UCAtkLL_v3 antibody. Insome embodiments, the SEC purification affects binding andneutralization properties. For example, SEC purified CH235UCA antibodyshows reduced neutralization and binding, whether or not the viruses areproduced in GnTI−/− cells. See FIG. 43 and FIG. 44 where Lot 48EML wasnot purified by SEC, and lot 170712PPF was purified by SEC (See FIGS. 41and 42 ).

Experiments in Example 13 are conducted with SEC purified antibodyunless noted otherwise. In some of the figures, and in Examples 13 thesize exclusion chromatography purified antibody is referred to aspurified antibody.

Example 13

Germline-Targeting and Reverse Engineering to Elicit CH235.12 LineageBNAbs

This example provides some strategies and non-limiting embodiments ofimmunogens to induce broad neutralizing antibodies, including CH235lineage of antibodies.

The ability to stimulate germline B cells that give rise to broadlyneutralizing antibodies (bNAbs) is a major goal for HIV-1 vaccinedevelopment. bNAbs that target the CD4-binding site (CD4bs) and exhibitextraordinary potency and breadth of neutralization are particularlyattractive to elicit with vaccines. Glycans that border the CD4bs andimpede the binding of germline-reverted forms of CD4bs bNAbs arepotential barriers to naïve B-cell receptor engagement. We usedpseudovirus neutralization as a means to identify Env modifications thatpermit native Env trimer binding to germline reverted CD4bs bNAbCH235.12 (VH1-46). Two mutations (N279K.G458Y), when combined withMan5-enrichment of N-linked glycans that are otherwise processed intocomplex glycans, rendered autologous CH0505TF Env highly sensitive toneutralization by CH235.12 UCA. These findings suggest a vaccine

Example 8 described a bnAb, CH235.12, which has ˜90% breadth, and usesVH1-46 chain. The deduced UCA for this lineages, CH235UCA does notneutralize wild type virus. Without bound by specific theory, virusmodifications that permit neutralization would be candidategermline-targeting immunogens. This information also suggestsreverse-engineering strategies to mature the response.

In some aspects, the goal was to identify Env modifications that permitneutralization by germline-reverted CD4bs bNAbs. In some embodiments,the hypothesis was that conversion of bulky complex-type glycans tosmaller Man5GlcNac2 glycoforms will reduce steric barriers to germlineBNAb binding without disrupting native Env conformation.

Previous work has explored glycan modifications and has shown thatdeletion of a subset of glycans surrounding the CD4bs is a feature thathas permitted binding and BCR activation by germline-reverted forms ofVRC01-class BNAbs. See McGuire et al., J Exp Med, 210:655-663, 2013;McGuire et al., Nat Comm 7:10618, 2016; Jardine et al., Science 340,711-716, 2013; Jardine et al., Science 349, 156-161, 2015; Jardine etal., Science 351, 1458-1463, 2016.

See also Zhou, T. et al. Cell Reports 19:719-732, 2017 where inter aliaCH0505TF; CH0505TF.gly4 (deleted N197, N276, N461, naturally lacksN362); CH0505TF.gly3.197; CH0505TF.gly3.276; CH0505TF.gly3.461 werestudied. Man5-enriched versions of these viruses were not neutralized byCH235UCA. Man5-enriched CH0505TF was highly sensitive to CH235intermediates. Glycan deletion did not improve neutralization by CH235intermediates

Induction of VH1-46 utilizing CD4 binding site (CD4bs) ANC131,CH235-class broadly reactive neutralizing antibodies (bnAbs) isdesirable because the affinity matured antibodies of this class arequite broad and potent, are not autoreactive, nor have long HCDR3regions, and, in the case of CH235, do not have difficult to induceinsertions or deletions that need to occur en route to bnAb breadth(Cell 165: 449-463, 2016). However, there are only a few of these bnAbsdescribed and only one bnAb lineage isolated from the time of acuteinfection to bnAb breadth, CH235 lineage (Cell 165: 449-463, 2016).Moreover, Envs that bind to the CH235 UCA at high affinity have not beenavailable. Here, we show that deletion of certain glycans and inclusionof a G458Y mutation creates a CH505 M5 Envelope from a low affinitybinding Env to a high affinity binding Env for the CH235 UCA.

Effect of Affinity of Immunizing Antigens on Induction of GerminalCenter (GC) Responses

The affinity of stimulating antigens has a profound effect on theoutcome of the germinal center response. High affinity antigens canprevent a B cell from staying in the germinal center, and promote rapidmaturation of a B cell to a short-lived plasma cell (Journal of Exp.Med. 203: 1081, 2006). Recent data suggest that affinities from highmicroM to low nM can activate bnAb precursors, but the key is what theaffinity of sequential Env immunogens must be to retain stimulated bnAbB cell lineages in the germinal center. To this end, we have selectedand produced at the Duke Human Vaccine Institute (DHVI) CGMP facilitythe M5 gp120 that has an apparent affinity for the CH235 UCA of 4.6microM while the mature CH235 bnAb has an affinity of 8.0 nM for the M5gp120 Env (FIG. 59F). Thus, in some embodiments the M5 gp120 could be a“low affinity” immunogen to determine its effectiveness in initiatingCH235-like VH1-46 CD4bs antibody B cell lineages.

For additional immunization strategies see also FIG. 58 and accompanyingdescription.

Design of the CH505 M5 G458Y Stabilized SOSIP Trimer that Targets theCH235 UCA at Various Affinity

To design an additional “high affinity” immunogen capable of binding tothe CH235 UCA at nM affinity, mutations that might increase binding ofthe CH235 UCA to Env based on the CH235-Env co-crystal structure (Cell165: 449-463, 2016) were studied. The CH505 M5 Env expressed as astabilized (4.1) SOSIP trimer bound to the CH235 UCA with an apparent Kdof 231 nM (FIG. 46 ). It was found that the G458Y Env mutation increasedthe apparent affinity of the CH235 UCA to 89 nM, and the M5 virus withthis mutation was able to be neutralized by the CH235 UCA (FIG. 46 ).Study of this Env with CH235 UCA using CryoEM demonstrated that 458Yinteracted with W50 in the CH235 UCA, and thus was a stabilizingmutation for this interaction (FIGS. 54, 55 ).

Non-limiting examples of neutralization of envelopes comprising G458Mutare shown in FIG. 89 . In some embodiments envelopes comprising G458C orG458L mutation will be analyzed further in various assay, including SPR,immunogenicity and so forth. In some embodiments these envelopes alsocomprise N279X, wherein examples of X are show in FIG. 90 .

Non-limiting examples of neutralization of envelopes comprising aminoacids other than lysine (K) at Env position 279 are shown in FIG. 90 .

Multimerization of CH505 M5 G458Y Env

It remains to be determined if multimerization of Env immunogens will berequired for optimal immunogenicity. Multimerization strategies are morecomplicated and purification of trimer multimers will requireconsiderable pre-production work. We have developed methods forexpressing and purifying the CH505 M5 G458Y Env. FIG. 60A shows a modelof the hexamer of M5 SOSIP trimers expressed on a ferritin scaffold.FIG. 60B shows purified CH505 M5 trimers after analysis innegative-stained EMs with class averages of the hexamer in multipleorientations. Purification plans for a trimer multimer have initiated.In the pre-production studies we will first develop two CH505 M5 G458Yresearch cell banks in the DG44 cell line, one expressing trimers, andone expressing hexamers. The process described above for trimers will bethe starting point for purification of CH505 M G458Y trimers. The choiceof which Env to move forward (trimer vs. hexamer of trimers) will bebased on ongoing immunogenicity studies in CH235 UCA knock-in mice,rabbits or macaques.

Any of the immunogens of the invention could be tested for Ca2+ flux ina suitable cell line comprising a desired antibody, e.g. but not limitedto CH235UCA2.

Animal Studies

The immunogens of the invention could be studied in various animalmodels. In some embodiments, the immunogenicity will be studied in ananimal model comprising CH235UCA VH and/or VL chains knocked into ananimal, e.g. a mouse. Any suitable animal could be used includingrabbits, mice, and non-human primates.

In one example, CH235UCA knock in mice are immunized as follows:

Group 1: CH505 M5 SOSIPsG458Y grown in GnTI−/− cells (×4)×5 mice

Group 2: M5 gp120delta8 (×4)×4 mice

Adjuvant for both groups is GLA-SE. The immunogenicity in these animalswill be analyzed by any suitable assay including neutralization, ELISA,etc.

Animal studies wherein the immunogens of the invention are administeredas mRNA, for example but not limited as modified mRNAs formulated inLNPs, or self-replicating mRNAs formulated in LNPs.

Additional Sequences

Table 29 shows a summary of the sequence Evolution of CH235 Lineage:SHM, Timing, and Conformity of CH235-Lineage Development from UCA toAntibody with 90% Breadth. V_(H)-gene mutability accounts for themajority of positional conformity of CH235 lineage. (SEQ ID NOS 314-323,respectively). SEQ ID NOS 314-323 are included in the Sequence Listingwhich is submitted electronically herewith in ASCII format and is herebyincorporated by reference in its entirety and forms part of thespecification. See also FIG. 43C of U.S. Provisional Application No.62/511,226 filed May 25, 2017 and U.S. Provisional Application No.62/565,952 filed Sep. 29, 2017.

TABLE 29 Name SEQ ID NO IGHV1-46*01 314 CH235 315 CH235.9 316 CH235.12317 1B2530 318 8ANC131 319 IGHV1-2*02 320 VRC01 321 VRC-CH31 322VRC-PG04 323

Table 30 shows a summary of CH235 Lineage: Sequences and NeutralizationFingerprint Dendrogram. Sequences (SEQ ID NOS 324-385, respectively) andantibodies isolated from 17 time points from 6 to 323 weekspost-transmission and comparison of mutation patterns to other IGHV1-46(1B2530 and 8ANC131) and IGHV1-2 (VRC01, VRC-CH31 and VRC-PG04) derivedbroadly neutralizing antibodies. IGHV1-46*01 is used as reference forIGHV1-46 derived antibodies and IGHV1-2*02 is used as reference for thethree VRC01-class antibodies. SEQ ID NOS 324-385 are included in theSequence Listing which is submitted electronically herewith in ASCIIformat and is hereby incorporated by reference in its entirety and formspart of the specification. See also FIG. 48A of U.S. ProvisionalApplication No. 62/511,226 filed May 25, 2017 and U.S. ProvisionalApplication No. 62/565,952 filed Sep. 29, 2017.

TABLE 30 Name SEQ ID NO IGHV1-46*01 324 UCA 325 122w14 326 39w20 32743w20 328 66w20 329 6w20 330 3w20 331 35w22 332 18w22 333 64w22 33416w22 335 30w22 336 15w22 337 13w22 338 65w22 339 20w22 340 10w22 34148w22 342 82w22 343 14w22 344 31w22 345 11w22 346 2w22 347 118w30 348117w30 349 132w30 350 100w41 351 90w41 352 74w41 353 70w41 354 47w41 3554w41 356 7w41 357 63w41 358 99w41 359 80w41 360 67w41 361 CH235 362CH236 363 CH239 364 CH240 365 CH241 366 28w53 367 24w53 368 1w53 369124w66 370 49w66 371 CH235.6 372 CH235.7 373 CH235.8 374 CH235.9 375CH235.10 376 CH235.11 377 CH235.12 378 CH235.13 379 1B2530 380 8ANC131381 IGHV1-2*02 382 VRC01 383 VRC-CH31 384 VRC-PG04 385

Table 31 shows a summary of sequence Similarity Between VH1-2 and VH1-46Broadly Neutralizing Antibodies and Mutability of Germline Genes. Aminoacid alignment of 8ANC131 (SEQ ID NO: 387) and CH235 (SEQ ID NO: 388) tothe IGHV1-46 (SEQ ID NO: 386) germline gene was performed. SEQ ID NOS386-388 are included in the Sequence Listing which is submittedelectronically herewith in ASCII format and is hereby incorporated byreference in its entirety and forms part of the specification. See alsoFIG. 50A of U.S. Provisional Application No. 62/511,226 filed May 25,2017 and U.S. Provisional Application No. 62/565,952 filed Sep. 29,2017.

TABLE 31 Name SEQ ID NO IGHV1-46 386 8ANC131 387 CH235 388

Table 32 shows a summary of sequence probability distribution of thenumber of sharing mutation positions for each pair of antibodies (SEQ IDNOS 389-395, respectively, in order of appearance). SEQ ID NOS 389-395are included in the Sequence Listing which is submitted electronicallyherewith in ASCII format and is hereby incorporated by reference in itsentirety and forms part of the specification. See also FIG. 50B of U.S.Provisional Application No. 62/511,226 filed May 25, 2017 and U.S.Provisional Application No. 62/565,952 filed Sep. 29, 2017.

TABLE 32 Name SEQ ID NO Probability distribution of sharing mutationpositions 389 Probability distribution of sharing mutation positions 390Probability distribution of sharing mutation positions 391 Probabilitydistribution of sharing mutation positions 392 Probability distributionof sharing mutation positions 393 Probability distribution of sharingmutation positions 394 Probability distribution of sharing mutationpositions 395

Table 33 shows primers designed with the online Agilent Quikchangeprimer designer tool (www.thermofisher.com) (SEQ ID NOS 8-15,respectively, in order of appearance).

TABLE 33 SEQ ID Name Sequence NO CH235.9_(N30T)CGTGGCGTCTGGATACAACTTCACCGAC 8 TACTATATAC CH235.9_(D31T)CGTCTGGATACAACTTCAACACCTACTA 9 TATACACTGGGTGC CH235.9_(G62Q)GGTCGCACAGATTACGCACAGGCGTTTG 10 GGGA CH235.9_(G65Q)GATTACGCAGGGGCGTTTCAGGACAGAG 11 TGTCCA CH235.9_(A103E)GTTAGAAATGTGGGAACGGAGGGCAGCT 12 TGCTCCACTATG CH235.9_(G62Q/G65Q)GGTCGCACAGATTACGCACAGGCGTTTC 13 AGGACAGAGTGTCCA CH235.9_(S54R)GGATCGACCCTAGGGGTGGTCGCACAG 14 CH235.9_(A61S)GTGGTCGCACAGATTACTCAGGGGCGTTTG 15

Table 34 shows designed PCR primers. PCR amplifications performed with acommon 5′ primer II A (Clontech) and an Ig gene specific 3′ primer (SEQID NO: 16) using KAPA HIFI qPCR kit (Kapa Biosystems). PCR amplificationperformed with primers with 454 sequencing adapters

(454-RACE-F: 5′CCATCTCATCCCTGCGTGTCTCCGACTCAGAAGCAGTGGTATCAACGCAGAGT3′ (SEQ ID NO: 17); 454-IgG-R:5′CCTATCCCCTGTGTGCCTTGGCAGTCTCAGGGGGAAGACCGATGGGCCCTTGGTGG3′ (SEQ ID NO: 18)).

TABLE 34 SEQ Sequence ID NO 5′GGGGAAGACCGATGGGCCCTTGGTGG3′ 165′CCATCTCATCCCTGCGTGTCTCCGACTCAGAAGCAGTGG 17 TATCAACGCAGAGT3′5′CCTATCCCCTGTGTGCCTTGGCAGTCTCAGGGGGAAGAC 18 CGATGGGCCCTTGGTGG3′

What is claimed is:
 1. A recombinant HIV-1 envelope polypeptidecomprising HIV-1 envelope CH505 M5 with a mutation of the amino acid atposition 458, wherein the HIV-1 envelope CH505 M5 polypeptide is: agp120 envelope comprising SEQ ID NO:424, a gp120 delta 8 envelopecomprising all the consecutive amino acids immediately after the signalpeptide sequence MRVMGIQRNYPQWWIWSMLGFWMLMICNG of SEQ ID NO:407, a gp140envelope comprising SEQ ID NO:423, a gp140 envelope comprising all theconsecutive amino acids immediately after the signal peptide sequenceMPMGSLQPLATLYLLGMLVASVLA of CH505M5chim.6R.SOSIP.664v4.1_G458Y (SEQ IDNO:405), a gp140 envelope comprising all the consecutive amino acidsimmediately after the signal peptide sequence MPMGSLQPLATLYLLGMLVASVLAof CH505M5chim.6R.SOSIP.664v4.1 ferritin_G458Y (SEQ ID NO:408), a gp140envelope comprising all the consecutive amino acids immediately afterthe signal peptide sequence MPMGSLQPLATLYLLGMLVASVLA ofCH505M5chim.6R.DS.SOSIP.664(SEQ ID NO:409), a gp140 envelope comprisingall the consecutive amino acids immediately after the signal peptidesequence MPMGSLQPLATLYLLGMLVASVLA ofCH505M5chim.6R.SOSIP.664v5.2.8_G458Y (SEQ ID NO:410), a gp140 envelopecomprising all the consecutive amino acids immediately after the signalpeptide sequence MPMGSLQPLATLYLLGMLVASVLA ofCH505M5chim.6R.SOSIP.664v4.1avi_G458Y (SEQ ID NO:411), a gp140 envelopecomprising all the consecutive amino acids immediately after the signalpeptide sequence MPMGSLQPLATLYLLGMLVASVLA ofCH505M5chim.6R.SOSIP.664v4.1.1_G458Y (SEQ ID NO:412), a gp140 envelopecomprising the amino acid sequence of CH505M5chim.6R.SOSIP.664_G458Y(SEQ ID NO:426), a gp140 envelope comprising all the consecutive aminoacids immediately after the signal peptide sequenceMPMGSLQPLATLYLLGMLVASVLA of CH505M5chim.6R.SOSIP.664v4.2 (SEQ IDNO:243), a gp145 envelope comprising all the consecutive amino acidsimmediately after the signal peptide sequenceMRVMGIQRNYPQWWIWSMLGFWMLMICNG of SEQ ID NO:187, or a gp160 envelopecomprising SEQ ID NO:422; wherein the amino acid at position 458 is atyrosine (Y), a phenylalanine (F), a tryptophan (W), an arginine (R), acysteine (C), a leucine (L), a lysine (K), a serine (S), an asparticacid (D), a glutamic acid (E), a methionine (M), a glutamine (Q), ahistidine (H), or an asparagine (N).
 2. The recombinant HIV-1 envelopepolypeptide of claim 1, wherein the HIV-1 envelope CH505 M5 polypeptideis a gp140 HIV-1 CH505 M5 polypeptide comprising: all the consecutiveamino acids immediately after the signal peptide sequenceMPMGSLQPLATLYLLGMLVASVLA of CH505M5chim.6R.SOSIP.664v4.1_G458Y (SEQ IDNO:405), all the consecutive amino acids immediately after the signalpeptide sequence MPMGSLQPLATLYLLGMLVASVLA ofCH505M5chim.6R.SOSIP.664v4.1 ferritin_G458Y (SEQ ID NO:408), all theconsecutive amino acids immediately after the signal peptide sequenceMPMGSLQPLATLYLLGMLVASVLA of CH505M5chim.6R.DS.SOSIP.664(SEQ ID NO:409),all the consecutive amino acids immediately after the signal peptidesequence MPMGSLQPLATLYLLGMLVASVLA ofCH505M5chim.6R.SOSIP.664v5.2.8_G458Y (SEQ ID NO:410), all theconsecutive amino acids immediately after the signal peptide sequenceMPMGSLQPLATLYLLGMLVASVLA of CH505M5chim.6R.SOSIP.664v4.1avi_G458Y (SEQID NO:411), all the consecutive amino acids immediately after the signalpeptide sequence MPMGSLQPLATLYLLGMLVASVLA ofCH505M5chim.6R.SOSIP.664v4.1.1_G458Y (SEQ ID NO:412), the amino acidsequence of CH505M5chim.6R.SOSIP.664_G458Y (SEQ ID NO:426), or all theconsecutive amino acids immediately after the signal peptide sequenceMPMGSLQPLATLYLLGMLVASVLA of CH505M5chim.6R.SOSIP.664v4.2 (SEQ IDNO:243), wherein the amino acid at position 458 is a tyrosine.
 3. Arecombinant HIV-1 envelope polypeptide of claim 1, wherein therecombinant HIV-1 envelope polypeptide is enriched for Man5 glycoformsof N-linked glycans.
 4. A nucleic acid encoding the recombinant HIV-1envelope polypeptide of claim
 1. 5. A recombinant trimer comprisingthree identical recombinant HIV-1 envelope polypeptides from claim
 2. 6.An immunogenic composition comprising the recombinant trimer of claim 5and a carrier.
 7. An immunogenic composition comprising the nucleic acidof claim 4 and a carrier.
 8. The composition of claim 6, furthercomprising an adjuvant.
 9. The nucleic acid of claim 4, wherein thenucleic acid is operably linked to a promoter inserted in an expressionvector.
 10. A method of inducing an immune response in a subject, themethod comprising administering a composition in an amount sufficient toinduce an immune response, wherein the composition comprises: one ormore of the recombinant HIV-1 envelope CH505 M5 polypeptides of claim 1,wherein the amino acid at position 458 is a tyrosine (Y); a nucleic acidencoding one or more of the HIV-1 envelope CH505 M5 polypeptides ofclaim 1, wherein the nucleic acid encodes a tyrosine (Y) at amino acidposition 458; or any combination thereof.
 11. The method of claim 10,wherein one or more of the recombinant HIV-1 envelope CH505 M5 G458Ypolypeptides is enriched for Man5 glycoforms of N-linked glycans. 12.The method of claim 10, further comprising administering a compositioncomprising one or more recombinant HIV-1 envelope polypeptidescomprising HIV-1 envelope CH505 M5, a nucleic acid encoding one or moreof the HIV-1 envelope polypeptides CH505 M5, or any combination thereof,wherein the HIV-1 envelope CH505 M5 polypeptide is: a gp120 delta 8envelope comprising all the consecutive amino acids immediately afterthe signal peptide sequence MRVMGIQRNYPQWWIWSMLGFWMLMICNG of SEQ IDNO:151, a gp160 sequence comprising all the consecutive amino acidsimmediately after the signal peptide sequenceMRVMGIQRNYPQWWIWSMLGFWMLMICNG of SEQ ID NO:163, or a gp145 sequencecomprising all the consecutive amino acids immediately after the signalpeptide sequence MRVMGIQRNYPQWWIWSMLGFWMLMICNG of SEQ ID NO:187.
 13. Themethod of claim 12, wherein one or more of the recombinant HIV-1envelope CH505 M5 polypeptides is enriched for Man5 glycoforms ofN-linked glycans.
 14. The method of claim 12, further comprisingadministering a composition comprising a recombinant HIV-1 envelopeCH505 T/F polypeptide, a nucleic acid encoding one or more of the HIV-1envelope polypeptides CH505 T/F, or any combination thereof, wherein theHIV-1 envelope CH505 TF polypeptide comprises: all the consecutive aminoacids immediately after the signal peptide sequenceMPMGSLQPLATLYLLGMLVASVLA in CH505TFchim.6R.SOSIP.664 (SEQ ID NO:234),all the consecutive amino acids immediately after the signal peptidesequence MPMGSLQPLATLYLLGMLVASVLA in CH505TFchim.6R.DS.SOSIP.664 (SEQ IDNO:238), all the consecutive amino acids immediately after the signalpeptide sequence MPMGSLQPLATLYLLGMLVASVLA inCH505TFchim.6R.SOSIP.664V4.1 (SEQ ID NO:260) all the consecutive aminoacids immediately after the signal peptide sequenceMPMGSLQPLATLYLLGMLVASVLA in CH505TFchim.6R.SOSIP.664V4.2 (SEQ IDNO:261).
 15. The method of claim 14, wherein one or more of therecombinant HIV-1 envelope CH505 T/F polypeptides is enriched for Man5glycoforms of N-linked glycans.
 16. The method of claim 10, wherein thecomposition further comprises a carrier.
 17. The method of claim 10,wherein the composition further comprises an adjuvant.
 18. The method ofclaim 10, further comprising administering an agent which modulates hostimmune tolerance.
 19. The method of claim 10, wherein the polypeptideadministered is multimerized in a liposome or nanoparticle.
 20. Themethod of claim 10, wherein the polypeptide administered is in the formof a recombinant trimer comprising three identical recombinant HIV-1envelope polypeptides.